Systems and methods for handling cryogenic charged particle samples - Patents.com

Abstract

To provide a cryogenic charged particle (CCP) sample handling and storage system used for storing and handling a cryogenic sample used in a charged particle microscope, such as a cryogenic electron microscope sample used in a cryogenic transmission electron microscope.SOLUTION: A system comprises a storage device for storing a plurality of CCP samples, and a charged particle device (CPA) such as cryo-TEM located away from the storage device. The system further comprises a transport device that is releasably connectable to the storage device and is also releasably connectable to the CPA. The transfer device is configured to take CCP samples from the plurality of CCP samples when connected to the storage device, and is configured to transfer the CCP samples from the transfer device to the CPA when connected to the CPA.SELECTED DRAWING: Figure 7b

Description

[Technical field]

[0001] overview The present invention relates to systems and methods for handling cryogenic charged particle (CCP) samples, including storage devices, transport devices, and charged particle instruments (CPAs) such as charged particle microscopes (CPMs).

[0002] Biology is the natural science that studies life and living organisms, including their physical structure, chemical processes, molecular interactions, physiological mechanisms, expression, and evolution.

[0003] Cell biology is the branch of biology that studies the structure and function of cells, the fundamental units of life. Cell biology is concerned with the physiological properties, metabolic processes, signaling pathways, life cycle, chemical composition, and interactions of cells with their environment. In cell biology, molecular recognition between macromolecules governs all of the most sophisticated processes within the cell. The most common macromolecules include biopolymers (nucleic acids, proteins, carbohydrates, and lipids), as well as large non-polymers (such as lipids and macrocycles).

[0004] Many researchers are interested in studying macromolecular complexes in their native environment at high resolution to reveal their structural dynamics and interactions. Charged particle microscopy can be used for this purpose.

[0005] Charged particle microscopy, especially in the form of electron microscopy (EM), is a well-known and increasingly important technique for imaging microscopic objects.Historically, the basic type of electron microscope has evolved into many well-known instruments, such as transmission electron microscope (TEM), scanning electron microscope (SEM) and scanning transmission electron microscope (STEM), and also into various auxiliary devices, such as the so-called "dual beam" tool (e.g., FIB-SEM), which additionally uses a focused ion beam (FIB) to "machine", allowing ion beam milling or ion beam induced deposition (IBID).Those skilled in the art will be familiar with different types of charged particle microscopy.

[0006] In an SEM, a scanning electron beam is directed at a sample, causing the emission of "auxiliary" radiation from the sample in the form of secondary electrons, backscattered electrons, X-rays, and cathodoluminescence (infrared, visible, and / or ultraviolet photons). One or more components of this emitted radiation can be detected and used for sample analysis.

[0007] In a TEM, a beam of electrons is passed through a sample to form an image from the interaction of the electrons with the sample as the beam passes through the sample. This image is magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device (CCD). The scintillator converts the primary electrons in the microscope into photons that the CCD can detect.

[0008] EM offers many methods for the study of biological samples: conventional TEM is used to study the overall morphology of biological samples, electron crystallography or single particle analysis is dedicated to the study of proteins and macromolecular complexes, and (cryo)electron tomography or cryo-EM in vitreous sections (CEMOVIS) aims at the structure of cellular organelles and molecules. In cryo-EM and CEMOVIS, the sample is preserved by rapid freezing using vitrification techniques and observed in a cryo-TEM. CEMOVIS adds the cryo-sectioning of the sample, which can be performed using cryo-FIB techniques.

[0009] Preparing biological samples for study in analytical instruments is often time-consuming and labor-intensive. Preparing a cryo-EM specimen involves, for example, taking an aqueous sample of biological material (usually a purified protein complex), applying it to a supporting tissue (grid), reducing it into a layer as thin as possible (100-800 Å depending on the size of the biomolecule), and freezing this layer quickly enough to prevent the water from crystallizing. Many aspects of this process of preparing biological samples are problematic.

[0010] Once a Cryo-EM sample has been prepared, it must be stored and handled in the proper conditions for later use in charged particle microscopy. For this purpose, multiple Cryo-EM samples can be stored in individual grid boxes. These grid boxes can then be placed in conical Falcon tubes and stored in long-term liquid nitrogen storage Dewars.

[0011] If necessary, remove the Falcon tube from the Dewar and remove the correct grid box corresponding to the desired sample from the Falcon tube.

[0012] In order to get the desired sample into the cryo-EM, various methods can be applied.

[0013] In a first embodiment, a cryo-transfer holder can be used. This is a liquid nitrogen holder designed for frost-free transfer of samples to a transmission electron microscope (TEM) at liquid nitrogen temperatures. The sample is removed from the grid box and manually placed in the cryo-transfer holder. The cryo-transfer holder can then be connected to the TEM. The cryo-transfer holder holds the sample in place in the TEM during acquisition. Placement of the cryo-transfer holder in the TEM has proven to be cumbersome, as there is a relatively high failure rate of cryo-holder insertion into the microscope. A significant amount of sample drift occurs for 20 minutes after connecting the cryo-transfer holder. Furthermore, the cryo-transfer holder limits the throughput of samples in the TEM, as the cryo-transfer holder requires manual refilling of liquid nitrogen every 12 hours and the cryo-cycle of the microscope required after 4-5 insertions is long, at 4 hours.

[0014] In a second embodiment, the sample is loaded into the TEM using a cryogenic sample handling robot. Such a sample handling robot is available, for example, from Thermo Scientific TM A product marketed by Sigma-Aldrich is known as AutoLoader. The AutoLoader is a module that is installed as part of a TEM. Samples are manually placed in the AutoLoader Cartridge. These AutoLoader Cartridges are stored in a cryogenic storage, such as a Dewar. When required, the Cartridge is removed from the Dewar and placed in the AutoLoader Module. The desired sample is then removed from the Cartridge by a handling robot, which transfers the sample to a sample holder in the TEM. The sample can then be imaged using the TEM. This known system provides good results, especially in terms of sample transfer quality and screening throughput, but there is room for improvement on other levels, for example improvements in the loading of samples into the cartridges, the complexity, the ease of installation and maintenance, and the associated costs.

[0015] From the above, it therefore follows that there is a desire for an improved sample storage and handling system that provides an improved user experience for cryogenic sample storage, cryogenic sample transport, and the study of cryogenic samples in a charged particle microscope or similar device.

[0016] To this end, the present disclosure provides a cryogenic charged particle (CCP) sample processing and storage system. The handling and storage system may be configured to handle and store, for example, cryo-electron microscopy samples.

[0017] The system as defined herein includes a storage device for storing a plurality of CCP samples, the storage device configured to store the CCP samples, such as Cryo-EM samples provided on a grid, under cryogenic conditions.

[0018] The system further comprises a charged particle apparatus (CPA) at a location remote from the storage device. The distance between the CPA and the storage device is such that direct transfer of the sample between the storage device and the CPA is not possible. Therefore, the system further comprises a transfer device configured to be movable between the storage device and the CPA.

[0019] The transfer device is releasably connectable to the storage device. This means that the transfer device can be connected and disconnected to the storage device. The transfer device is further releasably connectable to the CPA, so that the transfer device can be connected and disconnected to the CPA. In the disconnected state of the transfer device, the transfer device is movable between the storage device and the CPA. In the connected state of the transfer device, sample transfer is possible between the transfer device and the device to which it is connected. Thus, the transfer device can be configured to obtain a CCP sample from a plurality of CCP samples when connected to the storage device. Furthermore, the transfer device can be configured to transfer a CCP sample from the transfer device to the CPA when connected to the CPA.

[0020] This allows a single transfer device to be connected to a storage device to retrieve a sample, transport the transfer device with the sample to a CPA, such as a TEM, and load the sample into the CPA. Transfer from the transfer device to the CPA can include transfer to a sample holder (i.e., sample stage) that is part of the CPA.

[0021] Thus, the sample storage and handling system, and in particular the transfer device defined herein, allows samples stored under cryogenic conditions to be quickly and easily collected into a charged particle instrument, such as a TEM, for study. The collection of the sample (i.e., retrieval from the storage device), and the drop-off of the sample (i.e., transfer to the CPA) can be performed semi-automatically, i.e., without a human operator actually handling the sample. The transfer from the storage device to the CPA can be performed manually, i.e., by a human operator carrying the transfer device from the storage device to the CPA.

[0022] It should be noted that the storage and handling system may be configured to return, in other words, the transfer device is configured to acquire the sample at the CPA and transfer the sample from the transfer device to the storage device, which increases the versatility of the storage and handling system as defined herein.

[0023] It follows that the invention described herein provides an improved charged particle storage and handling system, which allows samples such as Cryo-EM samples to be easily and safely stored, transported to a charged particle device such as a (Cryo-)TEM, and returned to the storage device. Thus, an improved storage and handling system is obtained, thereby achieving the objectives defined herein.

[0024] Advantageous embodiments are described below.

[0025] In one embodiment, the system comprises at least one further CPA. The transfer device is configured to be releasably connectable to the further CPA, and thus the transfer device can also be connected and disconnected to the further CPA. In the disconnected state of the transfer device, the transfer device is movable between the storage apparatus and the CPA and the at least one further CPA. In the connected state of the transfer device, sample transfer is possible between the transfer device and the apparatus to which the transfer device is connected, such as the further CPA. Thus, the transfer device can be configured to transfer the CCP sample from the transfer device to the further CPA when connected to the further CPA. Thus, a single transfer device can be used in a system in which multiple CPAs are present. This central approach to the storage and handling system ensures that each of the multiple CPAs does not require a specific dedicated transfer mechanism, as for example when each of the CPAs is equipped with an AutoLoader module. In this way, the system can be constructively simple and therefore relatively inexpensive.

[0026] In one embodiment, the transfer device comprises a transfer mechanism configured to obtain the CCP sample from the storage device when connected to the storage device and deliver the CCP sample to the CPA when connected to the CPA. The transfer mechanism ensures a safe and reliable transfer between the apparatus of the system and the transfer device. No manual or manual sample handling is required to transfer the sample from the storage device to the transfer device. Additionally, no manual or manual sample handling is required to transfer the sample from the transfer device to the CPA.

[0027] The transfer mechanism may, for example, comprise a gripper, which may be configured to grip a CCP sample, in particular a cryo-EM sample.

[0028] The CCP samples used in the system may comprise EM grids. These grids themselves are known to those skilled in the art and may comprise small (a few millimeters) copper discs with a fine mesh with carbon foil on top. The grids may also be made of other materials. A liquid matrix layer containing the biological sample to be studied is applied to one side of the sample grid and then vitrified using a cryogenic cooling process known per se to those skilled in the art. The vitrified samples can then be stored in a storage device. A human operator can manually load the samples into the storage device. In one embodiment, that is the only direct human sample manipulation that needs to be used in the system.

[0029] Manually loading the samples into the storage device may include manually loading individual samples into a sample cassette. The storage device may include at least one cassette for storing at least a portion of the plurality of CCP samples. A human operator may place a desired sample into such a cassette and then place the cassette into the storage device. Of course, in that case, the transfer device is configured to retrieve the sample from the cassette.

[0030] In one embodiment, the transfer device is configured to be transferred from the storage device to the CPA by a human operator. In this manner, the system is relatively flexible as it allows the storage device and the (at least one) CPA to be positioned in a suitable location without the devices having to be positioned in close proximity to each other.

[0031] The storage device may be part of a first work station. The first work station comprises a human operator desk at which a human operator can sit or stand. The human operator can manually store or retrieve a plurality of samples from the storage device. The human operator can additionally or alternatively connect a transfer device to the storage device. The transfer device can be docked to a part of the first work station for that purpose.

[0032] In one embodiment, the storage apparatus and the CPA each comprise a first docking member and the transfer device comprises a further (second) docking member configured to mate with the first docking member. In this way, it is relatively easy to establish a connection between the transfer device and the storage device, disconnect the transfer device from the storage device, move the transfer device to the CPA, and then connect the transfer device to the CPA and transfer the sample from the transfer device to the CPA.

[0033] In one embodiment, each of the first docking members comprises a first valve and the further docking members comprise a further valve, the valve being configured to be closed in the detached state and the valve being configured to be openable in the docked state. In this manner, cryogenic conditions and / or other conditions (e.g., pressure, etc.) can be maintained in the storage apparatus, the transfer device, and the CPA. The transfer device can be connected to the storage apparatus by the docking member, and the valve can be opened such that the transfer space of the transfer device is fluidly connected to the storage space of the storage apparatus. Sample transfer can then be performed by fluid connection between the storage apparatus and the transfer device.

[0034] In one embodiment, the storage device comprises a cryogenic storage chamber. The storage chamber can be fluidly connected to the transfer device, in particular to the transfer space thereof. The storage chamber can be directly connected to the transfer space in the connected state of the transfer device. An intermediate chamber may also be provided. In that case, sample transfer can take place from the storage chamber to the intermediate chamber and then transfer can take place from the intermediate chamber to the transfer device. The storage device may also comprise a transfer mechanism. The storage device transfer mechanism may be configured to cooperate with the transfer mechanism of the transfer device, so that an effective sample transfer between the storage device and the transfer device can take place.

[0035] The storage device may comprise an operator input device and a control unit. In an embodiment, the input device may be part of the first work station. The operator input device may be configured to select the CCP sample to be transferred by a human operator. The control unit is configured to control, in particular in an automated manner, the storage device and the transfer device for transferring the selected CCP sample from the storage device to the transfer device. This increases the reliability of the system by allowing correct labeling and subsequent handling of the samples.

[0036] According to one aspect, there is provided a storage device for storing a plurality of cryogenic charged particle (CCP) samples for use in a CCP sample handling system as defined herein, the storage device configured to be releasably connectable to a transfer device and to enable transfer of a sample of the plurality of CCP samples from the storage device to the connected transfer device.

[0037] According to one aspect, there is provided a transfer device for transferring cryogenic charged particle (CCP) samples for use in a CCP sample handling system as defined herein, the transfer device being releasably connectable to a storage apparatus for storing a plurality of CCP samples and releasably connectable to a charged particle apparatus, the transfer device being configured to obtain a CCP sample from the plurality of CCP samples when connected to the storage apparatus, and configured to transfer a CCP sample from the transfer device to the CPA when connected to the CPA.

[0038] According to one aspect, there is provided a charged particle instrument (CPA) such as a charged particle microscope (CPM), e.g. a (Cyo-)TEM, for use in a charged particle (CCP) sample handling system as defined herein, the CPA configured to be releasably connectable to a transfer device of the system and to allow transfer of a sample from the transfer device to the connected CPA.

[0039] According to one aspect, a method for transferring a sample is provided. The method comprises the steps of providing a CCP sample handling system as defined herein. In this embodiment, the method comprises: - storing at least one CCP sample in said storage device; - connecting said transfer device to said storage device; - transferring the CCP sample from the storage apparatus to the transfer device; - removing said transfer device from said storage apparatus; - moving the transfer device to the charged particle apparatus (CPA), the CPA being located away from the storage apparatus; - connecting a transport device to a CPA; - transferring the CCP sample from the transfer device to the CPA.

[0040] The advantages of the method defined herein have been elucidated above. Advantageous embodiments are described below.

[0041] The step of transferring the sample can be performed mechanically. A mechanical transfer mechanism can be used to establish the transfer.

[0042] The step of transferring the sample may be performed automatically, meaning that no handling and / or manipulation of the sample by a human operator is required. However, it is conceivable that a human operator may initiate sample transfer, for example, by using a button, dial, knob, etc., or by entering a corresponding command into a user interface, such as a graphical user interface (GUI).

[0043] The steps of connecting and / or disconnecting can be performed by a human operator who physically connects the transfer device to the storage device or CPA. Some parts of the connecting and / or disconnecting may, in one embodiment, require the use of a docking mechanism, such as opening and closing a valve.

[0044] The step of moving the transfer device may be performed by a human operator physically moving the transfer device to or from the storage device and / or the CPA.

[0045] The methods and systems defined herein allow for safe and effective sample transfer, where a human operator is used to transfer samples between associated apparatus, and where more specialized transfer mechanisms allow for safe and reliable transfer from a transfer device to an apparatus, thus achieving the objectives as defined herein. [Brief description of the drawings]

[0046] The invention will now be elucidated in detail on the basis of exemplary embodiments and the accompanying schematic drawings. [Figure 1] 1 shows a longitudinal section of a charged particle microscope according to a first embodiment of the invention; [Diagram 2] 2 shows a longitudinal section of a charged particle microscope according to a second embodiment of the invention; [Diagram 3] 1 illustrates a prior art method of handling and storing samples. [Figure 4] 1 illustrates an embodiment of a sample handling and storage method and device as defined herein. [Diagram 5] 1 shows an embodiment of a transfer device for use in the sample handling and storage methods and apparatus defined herein. [Figure 6] 1 illustrates an embodiment of a transport device and a charged particle apparatus for use in the sample handling and storage methods and apparatus defined herein. [Figure 7a]FIG. 7a illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein. [Figure 7b] FIG. 7b illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein. [Figure 7c] FIG. 7c illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein. [Figure 7d] FIG. 7d illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein. [Figure 7e] FIG. 7e illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein. [Figure 7f] FIG. 7f illustrates various stages of a method for transferring a sample in a cryogenic charged particle (CCP) sample handling system as defined herein.

[0047] FIG. 1 (not to scale) is a very schematic depiction of an embodiment of a charged particle microscope M. More specifically, an embodiment of a transmission microscope M is shown, in this case a TEM / STEM (although in the context of the present invention, for example, an SEM (see FIG. 2) or an ion-based microscope may be equally useful). In FIG. 1, within a vacuum enclosure 2, an electron source 4 produces an electron beam B that propagates along an electron optical axis B', passes an electron optical illuminator 6, and serves to direct / focus the electrons onto a selected portion of a sample S (which may for example be (locally) thinned / planarized). A deflector 8 is also shown, which can be used to (among other things) effect a scanning motion of the beam B.

[0048] The sample S is held in a sample holder H, which can be positioned in multiple degrees of freedom by a positioning device / stage A that moves a cradle in which the holder H is (removably) mounted; for example, the sample holder H can be equipped with fingers that can be moved in the XY plane (among other things) (see Cartesian coordinate system in the depiction, usually movements parallel to Z and tilts about X / Y are also possible). Such movements allow different parts of the sample S to be illuminated / imaged / inspected by an electron beam B moving (in Z direction) along an axis B' (and / or a scanning movement can be performed as an alternative to electron beam scanning). If desired, an optional cooling device (not depicted) can be brought 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.

[0049] The electron beam B interacts with the sample S in such a way as to cause it to emit various types of "stimulated" radiation, including (for example) secondary electrons, backscattered electrons, X-rays, and light radiation (cathodoluminescence). If desired, one or more of these types of radiation can be detected with the aid of an analysis device 22, which can be, for example, a combination of a scintillator / photomultiplier tube or an EDX or EDS (energy dispersive X-ray spectroscopy) module, and in such a case an image can be constructed using essentially the same principles as in an SEM. However, alternatively or supplementarily, the electrons that traverse (pass) the sample S, emerge / emit from the sample S, and continue to propagate (substantially, but generally, deflected / scattered to some extent) along the axis B' can be studied. Such a transmitted electron flux enters an imaging system (projection lens) 24, which typically comprises various electrostatic / magnetic lenses, deflectors, collectors (stigmators, etc.), etc. In standard (non-scattering) TEM mode, this imaging system 24 can focus the electron flux onto a phosphor screen 26, which can be retracted / withdrawn (as diagrammatically indicated by arrow 26') if desired to bring it out of the path of axis B'. On the screen 26, an image (or diffractogram) of (a part of) the sample S is formed by the imaging system 24, which can be seen through a viewing port 28 located in a suitable part of the wall of the housing 2. The retraction mechanism of the screen 26 can for example be mechanical and / or electrical in nature and is not shown here.

[0050] As an alternative to viewing an image on screen 26, one can instead take advantage of the fact that the focusing depth of the electron flux leaving imaging system 24 is typically quite deep (e.g., on the order of one meter). As a result, various other types of analytical devices (such as those listed below) can be used downstream of screen 26. -TEM camera 30. At the position of camera 30, the electron flux can form a still image (or diffractogram) which can be processed by controller / processor 20 and displayed on a display device 14, such as a flat panel display. When not required, camera 30 can be retracted / retracted (as shown diagrammatically by arrow 30') to move the camera off axis B'. - STEM camera 32. The output from camera 32 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 32 as a function of X,Y. Camera 32 can also be an Electron Microscope Pixel Array Detector (EMPAD), but can comprise a single pixel, e.g., 20 mm in diameter, as opposed to the pixel matrix typically present in camera 30. Furthermore, camera 32 generally can be a 10 mm pixel array, e.g., ... 2 Much higher acquisition rates (e.g., 10 images / sec) 6 points / sec). Again, if not required, camera 32 may be retracted / pulled out (as indicated diagrammatically by arrow 32') out of the way of axis B' (such retraction may not be necessary, for example, in the case of a donut-shaped annular dark field camera 32, where a central hole may allow for bundle passage when the camera is not in use). As an alternative to imaging using the cameras 30 or 32, a spectroscopic device 34 can also be invoked, which can be, for example, an EELS module.

[0051] It should be noted that the order / position of components 30, 32, and 34 is not strict and many possible variations are possible. For example, spectroscopic device 34 could be integrated with imaging system 24.

[0052] In the illustrated embodiment, the microscope M further comprises a retractable X-ray Computed Tomography (CT) module, generally designated by the reference numeral 40. Computed tomography (also called tomographic imaging) uses electron sources and (diametrically opposed) detectors to interrogate a sample along different lines of sight to obtain an insightful view of the sample from different perspectives.

[0053] It should be noted that a controller (computer processor) 20 is connected to the various components shown via control lines (bus) 20'. This controller 20 may provide various functions such as synchronizing actions, providing set points, processing signals, performing calculations, and displaying messages / information on a display device (not shown). Needless to say, the controller 20 (shown diagrammatically) may be (partially) inside or outside the housing 2 and may have a unitary or composite structure, as desired. The controller, as shown in this embodiment, comprises a data processing device P configured for carrying out the method defined herein.

[0054] Those skilled in the art will understand that the interior of the housing 2 does not have to be kept in strict vacuum conditions. For example, in so-called "environmental TEM / STEM", a background atmosphere of a given gas is deliberately introduced / maintained inside the housing 2. Those skilled in the art will also understand that in practice, it may be advantageous, if possible, to restrict the volume of the housing 2 to essentially embrace the axis B', taking the form of a small tube (e.g. of dimensions of the order of 1 cm), transparent to the employed electron beam, but extending outwards to accommodate structures such as the electron source 4, the sample holder H, the screen 26, the cameras 30, 32, the spectrometer 34, etc.

[0055] Referring now to Figure 2, another embodiment of a charged particle device is shown. Figure 2 (not to scale) is a very schematic depiction of a charged particle microscope M, and more specifically shows an embodiment of a non-transmission microscope M, in this case an SEM (although in the context of the present invention, for example, an ion-based microscope may be equally useful). In the figure, parts corresponding to items in Figure 1 are indicated using the same reference numbers and are not considered separately here. The following parts (among others) are added to Figure 1: -2a: vacuum port, which can be opened to introduce / remove items (components, samples) to / from the interior of the vacuum chamber 2 or in addition thereto, for example, auxiliary devices / modules can be attached. The microscope M can be equipped with several such ports 2a if desired. - 10a, 10b: Schematically depicted lenses / optical elements in the illuminator 6. -12: A voltage source that allows the sample holder H or at least the sample S to be biased (floated) to a certain potential with respect to ground, if desired. -14: Display such as FPD or CRT. - 22a, 22b: (Segmented electron detector 22a, with a number of independent detection segments (e.g. quadrants) arranged around a central aperture 22b (allowing the passage of the beam B). Such a detector can be used, for example, to study the (angular dependence of) the flux of output (secondary or backscattered) electrons emerging from the sample S.

[0056] Here too, there is a controller 20. The controller is connected to a display 14, which in turn may be connectable to a data processing device P arranged to carry out the methods defined herein. In the embodiment shown, the data processing device P is a separate structure that does not form part of the controller or even part of the microscope P. The data processing device P may be local or cloud-based and is in principle not limited in location.

[0057] Charged particle instruments, and especially electron microscopes (EM), as shown in Figures 1 and 2, offer numerous methods for the study of biological samples: conventional TEM is used to study the overall morphology of biological samples, electron crystallography or single particle analysis is dedicated to the study of proteins and macromolecular complexes, and (cryo)electron tomography or Cryo-EM on vitreous sections (CEMOVIS) is aimed at organelles and molecular structure.

[0058] As indicated in the introduction, these biological samples can be preserved by flash freezing using vitrification techniques and then studied using cryo-EM techniques such as cryo-TEM. Cryosectioning of samples using cryo-FIB techniques can be part of the sample study.

[0059] The samples used in these studies must first be prepared and then stored. For this purpose, an aqueous sample of biological material (usually a purified protein complex) is taken, applied to a support structure (a grid), reduced in size to a very thin layer, and then this layer is frozen fast enough to prevent the crystallization of water. The sample is then prepared and stored for further handling.

[0060] As indicated at the beginning, one method for storing and handling samples relates to a so-called AutoLoader. An embodiment of this prior art will now be discussed with reference to Fig. 3. Fig. 3 shows a workflow for collecting samples S for transport to a charged particle microscope M, such as a cryo-TEM, using an AutoLoader AL and an associated NanoCab N cartridge. The AutoLoader AL is part of the microscope M. The NanoCab is loaded with multiple samples S, which are then transferred to the AutoLoader AL, which loads the samples into the TEM.

[0061] From left to right in the top row of FIG. 3, the preparation of a NanoCab N is shown. A number of samples S (i.e. biological specimens provided on a grid) are provided in a grid box G. The grid box G with the samples S is placed in a loading station 101. The loading station 101 is filled with liquid nitrogen 103 to keep the samples S and the grid box G at a desired low temperature. A cassette C is also provided in the liquid nitrogen 103. The samples S are manually transferred from the grid box G to the cassette C. Once the cassette is full, the Nanocab device N is connected to the loading station 101 and the cassette is provided in the Nanocab N. The Nanocab N device now contains a cassette C with the desired samples S, which is filled with liquid nitrogen to keep the samples S at a desired low temperature. Then, when or after the cassette C is introduced into the Nanocab device N, the liquid nitrogen is removed from the loading station so that the loading station 101 can return to room temperature.

[0062] The bottom part of Figure 3 shows that a Nanocab N with a cassette C with multiple samples S is connected to an Autoloader AL module, which is connected to or part of a microscope.

[0063] The Autoloader AL module comprises a cassette arm 113 and a sample arm 111. The interior of the Autoloader AL housing is kept at a desired low temperature and is configured to store cryogenic samples. The Autoloader AL module comprises two valve elements 115, 117. The first valve element 115 can provide connection to a connected Nanocab N. The second valve element 117 can provide connection to a microscope M.

[0064] The procedure for loading samples from the Nanocab N to the microscope M is as follows: The Nanocab N is connected to the Autoloader AL as shown in the lower part of Fig. 3. The valve 115 is opened and the cassette arm 113 reaches downwards to grip the cassette C with the sample S, then starts its upward movement to move the desired sample S in front of the sample arm 111. The valve 115 can be closed again.

[0065] The sample arm 111 can then collect the sample S, after which the cassette arm 113 moves the cassette C with the remaining samples further up and out of the way of the sample arm 111.

[0066] Valve 117 can then be opened and sample arm 111 moves the sample towards sample holder H of microscope M. Once sample S has been transferred to microscope M, sample arm 111 can return and valve 117 can be closed again. The sample can then be observed or manipulated.

[0067] As shown, this known Autoloader AL system offers excellent results, especially in terms of sample transfer quality and screening throughput. However, there is a desire to improve this system, especially with regard to sample loading, complexity, ease of installation and maintenance, and the associated costs.

[0068] To this end, the present invention provides a system for handling and storing cryogenic charged particle samples, such as cryo-EM samples. In general, the system includes a storage apparatus, a charged particle apparatus, and a transfer device configured to transfer the cryogenic sample from the storage apparatus to the charged particle apparatus.

[0069] FIG. 4 shows an embodiment of the storage device L. The top row of FIG. 4 shows how the samples S are loaded into the storage device L. For this purpose, the samples are provided in a grid box G. The grid box is placed in the housing 201 of the storage device L, which is partially filled with liquid nitrogen 203. A cassette C is also provided in the liquid nitrogen 203. The samples are then transferred manually from the grid box G to the cassette C (step 1). Once the samples S have been transferred, the cassette C remains in the housing of the storage device L (step 2). The cassette C may be moved to a separate storage location (not shown) of the storage device, for example by using a cassette arm 211 (see the bottom part of FIG. 4).

[0070] As shown in Figure 4, the cassettes C with the samples S are stored in a housing 201 of a storage device L. In this sense, the housing 201 with the liquid nitrogen 203 provides a cryogenic storage chamber for safe storage of the samples within the storage device.

[0071] When a required sample is required, the following procedure can be followed: First, a transfer device T as defined herein is provided, which is connected to a storage apparatus S.

[0072] The transfer device T comprises a housing 303 filled with gaseous nitrogen 303, in which the transferred sample S can be temporarily stored at an appropriate temperature. The transfer device T comprises a transfer arm 311 with a gripper 331. The gripper 331 can be used to collect the sample S. The gripper with the sample S can then be moved into the housing 303 of the transfer device T.

[0073] In the embodiment shown in Fig. 4, the storage L comprises a valve member 215. The transfer device T also comprises a valve member 315. The storage comprises a first docking member 221 configured to mate with a second docking member 321 of the transfer device T (shown diagrammatically). The storage may comprise a slot 221, into which the housing portion 321 of the transfer device may slide to provide a connection of the transfer device T and the storage. In this way, a translational docking is provided. Other docking mechanisms or ways of connecting the transfer device T to the storage L are also conceivable. It is advantageous if the docking member 321 of the transfer device T functions as a male connector 321 and the docking member 221 of the storage L functions as a female connector 221.

[0074] Once the transfer device T is connected (or docked) to the storage device L, the transfer of the sample S can take place. The valve 315 of the transfer device T is opened and the valve of the storage device 215 is also opened. The cassette arm 211 positions the desired sample S along the transfer arm 311 of the transfer device T. The transfer arm 311 moves in the housing 201 of the storage device L and takes the sample S from the cassette C using the gripper 331. The transfer device T is thus configured to acquire the sample S from the cassette C. The gripper 331 with the sample S is then moved inside the housing 303 of the transfer device T. Then all the valves 215, 315 are closed. The transfer device T can then be detached from the storage device L and moved to the charged particle device.

[0075] 4 shows the step of positioning the desired sample S using the cassette arm 211, which is performed mechanically, and in particular automatically, without the need for handling by a human operator. The human operator can select the desired sample through a user interface, but the movement, positioning, collection, and transfer are performed automatically. This limits the possibility of possible errors.

[0076] FIG. 5 shows the transfer device T in a cut state, with the sample S safely seated in the gripper 331 of the transfer arm 311. The sample is contained inside the housing 303, which is filled with gaseous nitrogen to keep the sample at the desired low temperature. The heat capacity of the gaseous cold nitrogen in the housing 303 is such that the transfer device maintains the sample at the desired temperature range for several minutes, for example 15 minutes. Additional cooling means may be provided on or inside the transfer device T to provide active cooling of the sample S in the housing 331. The transfer device T is transportable by a human operator. The dimensions of the transfer device T are chosen so that an average human operator can carry the transfer device from a first location to a second location, for example from a storage device L to a charged particle microscope M. The device has a length on the order of centimeters, decimeters or even meters. In practical embodiments, the device T has a length between 40 cm and 80 cm, although other dimensions are also conceivable. The weight of the device T may be on the order of one to several kilograms.

[0077] 6 shows a transfer device T connected to a microscope M. The microscope M includes a first docking member 421 that can mate with a second docking member 321 of the transfer device T. As mentioned above, the docking member 321 of the transfer device T can be formed by an external housing portion of the transfer device T. The microscope M can include a slot 421 that can receive the transfer device T, or at least the second docking member 321 thereof.

[0078] The microscope M has a valve member 415. Once the transfer device T is connected to the microscope M, or generally to the charged particle instrument CPA, the valves 315, 415 can be opened and the transfer arm 311 can move within the microscope to transfer the sample S to the holder H of the microscope M. This completes the transfer of the sample from the storage location to the charged particle instrument, such as the electron microscope M.

[0079] The transfer device T can be detached from the microscope M, so that while the microscope is inspecting the first sample S, transfer of the second sample S2 can occur.

[0080] Figures 7a-7f show diagrammatically a sample handling and storage system 500 as defined herein.

[0081] 7a shows that the system 500 comprises a storage device L for storing a number of samples S, S2. The storage device L may comprise a user input device 114, for example in the form of a conventional personal computer having a screen.

[0082] The system further comprises a charged particle device (CPA) M, such as a SEM, TEM, STEM and / or FIB. The CPA M is located at a distance from the storage device L. This means that in one embodiment, the CPA M is located at a distance of at least 50 cm from the storage device L. For example, the CPA may be located at a distance of one to several meters from the storage device. In practice, the distance between the storage device L and the charged particle device M is so large that a direct transfer from the storage device L to the device M is not possible and an intermediate transfer step is necessary. The advantage of such a system is that it is relatively flexible, since the storage device L and the charged particle device can be located at any desired location, even in different rooms with different environmental conditions.

[0083] To ensure that a safe and reliable sample transfer between the charged particle device CPA and the storage device L is possible, the system 500 comprises a transfer device T. The transfer device T can be handled by a human operator 600. The human operator 600 can carry the transfer device T to and from the storage device and to and from the charged particle device CPA M.

[0084] Turning now to FIG. 7b, it is shown that the transfer device T is releasably connectable to the storage L. As previously described with respect to FIG. 4, the transfer device is configured to acquire a CCP sample S from a plurality of CCP samples S, S2 when connected to the storage L. For this purpose, the transfer device T is connected to the storage L, for example by inserting the transfer device T into a slot present in the storage. The sample transfer described in FIG. 4 can then be performed. As shown in FIG. 7b and FIG. 7c, the sample is moved from the storage L to the transfer device T at a docking position of the transfer device T relative to the storage L. In one embodiment, the sample transfer from the storage L to the transfer device T is performed automatically, without the need for handling of the sample by a human operator.

[0085] Once the sample is in the transfer device T, the human operator can retrieve the transfer device T from the storage L. The transfer device T with the desired sample S can then be moved to a further location, such as a microscope. The human operator 600 can walk to the further location where the transfer device T is carried by the human operator 600.

[0086] Turning now to Figure 7e, it is shown that the transfer device T with the sample S is connected to a microscope M (or a charged particle device in general). A transfer mechanism of the transfer device T then transfers the sample S from the transfer device T to a sample holder H of the microscope M.

[0087] The sample may then be viewed and / or inspected under microscope M as described with respect to FIGS.

[0088] As shown in Figures 4-7, the transfer device T comprises a transfer mechanism 311, 331 configured to obtain the CCP sample S from the storage device L when connected to the storage device L, and to deliver the CCP sample to the CPA when connected to the CPA. In this way, a single mechanism can be used to transfer the sample between two external devices. The device should be arranged to cooperate with the transfer mechanism, but it is not necessary to have such a mechanism. Furthermore, the device may comprise additional means, including mechanical arms 211, valves 215, 415, etc., to optimize sample transfer and cooperate with the transfer mechanism 311, 331 of the transfer device.

[0089] In one embodiment, the transfer mechanism comprises a movable arm 311. The movable arm may be configured for translational movement. An outer end of the movable arm 311 may be provided with a gripper 331 configured to grip and release samples, including samples, particularly samples provided on sample grids. The sample grids may also be connected to other grid elements, which serve to facilitate sample handling. The grid elements may be, for example, C-clip rings (i.e. AutotGrid, Thermo Fisher Scientific) to which the sample grids may be attached. TM ) and a C-clip for fixing the sample grid within the C-clip ring. Of course, other grids are contemplated.

[0090] As shown in Figures 7a-7f, the storage device L comprises an operator input device 114 in the form of a computer and a control unit 220 connected to the operator input device 114 and configured to perform at least some functions of the storage device L or of the system 500, for example in connection with a transfer device T to the storage device L. The operator input device is configured in one embodiment for selecting, by a human operator 600, a CCP sample S, S2 to be transferred from the storage device to the charged particle microscope M. The control unit 220 is configured to control the storage device L and, in connection therewith, the transfer device T for transferring the selected CCP sample S from the storage device L to the transfer device T.

[0091] The storage device L including the operator input device 114 can form a first workstation, by which a human operator can quickly and reliably transfer prepared samples to the storage device L. The top of the housing 201 of the storage device L can match the desk function of the workstation. The top of the housing 201 and / or the desk function of the workstation can include a lid that can be opened and closed by a human operator, by which a grid box G (see FIG. 4, step 1) containing a sample S can be inserted into the storage device L and the sample grid S can be transferred to the container C. The lid can also be used to replenish the liquid nitrogen level 203 in the housing 201. The operator input device and / or the controller can be connected to the housing 201, so that information related to the housing 201 can be provided to the human operator. For example, the storage device can include several sensor elements, such as temperature sensors, liquid level sensors, etc., for monitoring the status of the storage device. The operator input device 114 can provide feedback to the user regarding one or more of these conditions.

[0092] It is further contemplated that the operator input device 114 may be used for feature labeling and tracking. In one embodiment, the operator input device 114 may be wirelessly connected to the charged particle instrument such that information regarding the sample S may be transferred to the charged particle instrument. The information may include sample input information, e.g., information entered into the operator input device 114 by a human operator when loading the sample S into the cassette C.

[0093] Above the system has been explained in more detail using exemplary embodiments. The desired protection is given by the appended claims.

Claims

1. 1. A cryogenic charged particle (CCP) sample handling system comprising: a storage device for storing a plurality of CCP samples; a charged particle apparatus (CPA) remote from the storage apparatus; a transfer device releasably connectable to the storage apparatus, the transfer device further releasably connectable to the CPA, the transfer device configured to obtain a CCP sample from the plurality of CCP samples when connected to the storage apparatus, and configured to transfer the CCP sample from the transfer device to the CPA when connected to the CPA; the storage device comprises at least one cassette for storing the plurality of CCP samples; the transfer device is configured to obtain a sample from the cassette; the transfer device having a housing filled with a gas refrigerant; CCP sample handling system.

2. 2. The CCP sample handling system of claim 1, wherein the transfer device comprises a transfer mechanism configured to obtain the CCP sample from the storage device when connected to the storage device, and to deliver the CCP sample to the CPA when connected to the CPA.

3. The CCP sample handling system of claim 2 , wherein the transport mechanism includes a gripper.

4. A CCP sample handling system according to any one of claims 1 to 3, wherein the transfer device is configured to be transferred from the storage device to the CPA by a human operator.

5. A CCP sample handling system according to any preceding claim, wherein the storage apparatus and the CPA each comprise a first docking member, and the transfer device comprises a further docking member configured to mate with the first docking member.

6. 6. The CCP sample handling system of claim 5, wherein each of the first docking members comprises a first valve and each of the further docking members comprises a further valve, the valves being configured to be closed in a detached position and configured to be openable in a docked position.

7. A CCP sample handling system according to any preceding claim, wherein the storage device comprises a cryogenic storage chamber.

8. A CCP sample handling system as described in any one of claims 1 to 7, wherein the storage device comprises an operator input device and a control unit, the operator input device configured to select a CCP sample to be transferred by a human operator, and the control unit configured to control the storage device and the transfer device for transferring the selected CCP sample from the storage device to the transfer device.

9. A transfer device for transferring cryogenic charged particle (CCP) samples for use in a CCP sample handling system according to any one of claims 1 to 8, said transfer device being releasably connectable to a storage device for storing a plurality of CCP samples, said transfer device being further releasably connectable to a charged particle device, said transfer device being configured to obtain a CCP sample from said plurality of CCP samples when connected to said storage device, and configured to transfer said CCP sample from said transfer device to said CPA when connected to said CPA; the storage device comprises at least one cassette for storing the plurality of CCP samples; the transfer device is configured to obtain a sample from the cassette; the transfer device having a housing filled with a gas refrigerant; Transport device.

10. A method of transferring a sample in a cryogenic charged particle (CCP) sample handling system according to any one of claims 1 to 8, comprising the steps of: providing said CCP sample handling system; storing at least one CCP sample in said storage device; connecting the transfer device to the storage device; charging a housing of the transfer device with a gaseous refrigerant; transferring the CCP sample from the storage apparatus to the transfer device; removing the transfer device from the storage apparatus; moving the transfer device to the charged particle apparatus (CPA), the CPA being located away from the storage apparatus; connecting the transport device to the CPA; transferring the CCP sample from the transfer device to the CPA.

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