NL2033291B1

NL2033291B1 - Device and Method for Cryogenic Electron Microscopy Sample Preparation

Info

Publication number: NL2033291B1
Application number: NL2033291A
Authority: NL
Inventors: Lamers Meindert; Fernandez Leiro Rafael
Original assignee: Academisch Ziekenhuis Leiden; Fundacion Del Sector Publico Estatal Centro Nac De Investigaciones Oncologicas Carlos Iii F S P Cnio
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2024-04-26
Also published as: WO2024080868A1

Abstract

Provided herein is a cryogenic electron microscopy sample preparation device that includes a sample support configured to receive a sample grid, a first applicator configured to comprise a first sample material, a second applicator configured to comprise a second sample material, a first gas outlet, a second gas outlet, at least one guard configured to move between a closed configuration and an open configuration, a cooling bath, a control unit, and an image capturing apparatus; and a method of preparing a sample for cryogenic electron microscopy employing the same.

Description

Device and Method for Cryogenic Electron Microscopy Sample Preparation

Field of the invention

[0001] The present disclosure relates to a cryogenic electron microscopy (cryo-EM) sample preparation device and cryo-EM sample preparation method, and particularly such devices and methods which facilitate performance of time-resolved cryo-EM experiments of target specimens under non-equilibrium conditions on a millisecond timescale.

Background

[0002] Cryogenic electron microscopy is a technique used to determine, among others, high resolution structures of biological macromolecules. Under traditional sample preparation techniques, an electron microscopy sample or specimen is prepared for imaging as follows. A fluid, which includes the sample or specimen to be imaged, is manually placed upon a metal grid sample support. This can be done, for example, by means of a pipette. The fluid wets and saturates the grid, filling the grid squares, and more particularly, the pores within the holey carbon supported by the grid. Excess fluid is then removed, and the sample support is cooled, typically by way of ‘plunge freezing’, such that the fluid containing the sample is vitrified. The sample support including the sample fluid is then stored until an imaging session is available at an electron microscope.

[0003] The excess fluid, which can impact the quality of the observed sample or specimen, is typically removed with filter paper that is used to “blot” away excess liquid from the sample grid that holds the sample or specimen. Such blotting processes involve pressing appropriate blotting papers against one or both surfaces of the sample support grid. However, these blotting processes often lead to variable results in relation to the sample fluid that is left on the sample grid. Some grid squares may contain little to no sample fluid while others may contain a relatively large volume of the sample fluid. This means that the fluid thicknesses of adjacent grid squares may vary considerably. Moreover, such variance across the sample grid can lead to variable image quality.

[0004] The final results of the sample preparation according to the blotting technique for removal of excess sample fluid may only be evaluated in the electron microscope, which is not suitable for high throughput analysis. As such, no quality control of prepared grids occurs until the grids reach the electron microscope for actual imaging.

[0005] It can be said that the success of the cryogenic electron microscopy technique is correlated with the quality of the sample introduced into the microscope. It is, therefore, important that thin layers of vitreous ice with heterogeneously distributed and randomly oriented particles be obtained on the sample grid.

However, it has been historically difficult to obtain samples with the requisite thin layers of vitreous water suitable for high resolution imaging from the standard blotting methodology given its aforementioned susceptibility to inconsistency; and this is not the sole disadvantage. Other disadvantages may include problems with preferred orientation, aggregation, adverse interactions with the air water interface, or the sample setup (e.g., blotting paper}, all of which can compromise the quality of the resultant sample(s). Not to mention, correcting for one or more of these disadvantages can add as much as seconds to sample grid preparation, which is slower than most biological processes that occur on a sub-second time scale and can be a hinderance for the performance of time resolved cryo-EM experiments of such biological processes, which are conducted on the millisecond time scale.

[0006] Different techniques have thus been developed in attempt to improve the reproducibility of grid preparation, such as pin printing thin layers of sample on the grids or spotting small drops of sample of self- wicking grids. Other alternatives, such as those described in “On-grid and in-flow mixing for time-resolved cryo-EM" by Klebl et al., have sought to develop time-resolved cryo-EM plunge-type freezers suitable for rapid mixing and spraying of small droplets onto holey carbon or self-wicking grids. WO 2018/020036 describes yet another electron microscopy sample preparation device comprising a gas outlet configured to direct a flow of gas towards a surface of an electron microscopy sample support to adjust fluid supported thereon, and in which a fluid sample is applied to the sample support either by a user with a pipette or with a fluid sample depositor operable to deposit the fluid sample on the sample support.

[0007] still, these alternative techniques are not without their own limitations, and further space for improvement remains, for example, in terms of applying the sample materials to the sample grid, optimizing the amount of time elapsed between composite sample formation (e.g., mixture of the sample materials) and vitrification of the composite sample, particularly on a sub-second time scale, and producing cryo-EM samples in a manner that is verifiably consistent and reproducible. Not to mention, there is also space to improve preliminary assessment of the quality of the prepared sample(s) in real-time, thus allowing for remediation of poor quality samples prior to electron microscopy imaging.

Summary of the invention

[0008] The present invention relates to a device for cryogenic electron microscopy (cryo-EM) sample preparation through which thin layers of vitreous ice with heterogeneously distributed and randomly oriented particles can be obtained on the sample grid with improved distribution. The cryo-EM sample preparation device described herein facilitates the rapid mixing and vitrification of two sample materials on a millisecond time scale. The instant cryo-EM sample preparation device thus makes it possible to obtain high resolution target specimens under non-equilibrium conditions on a millisecond timescale suitable for time-resolved cryo-electron microscopy. Additionally, the present invention relates to a cryo-EM sample preparation method.

[0009] A cryogenic electron microscopy (cryo-EM) sample preparation device is provided comprising a sample support configured tc receive a sample, a first applicator configured tc comprise a first sample material, a second applicator configured to comprise a second sample material, a first gas outlet, a second gas outlet, at least one guard configured to move between a closed configuration and an open configuration, a cooling bath, a control device, and an image capturing apparatus.

[0010] The provision of dual applicators is desirable because it facilitates the deposition of two sample materials directly onto the grid. This means that the sample material(s) can be mixed directly on the sample grid unlike the existing sample preparation techniques under which the sample materials are mixed prior to their application on the grid. This is advantageous because the amount of time elapsed between the formation of the composite sample and vitrification of the same in the cooling bath can be reduced. Stated in different terms, direct mixing of the sample material(s) on the grid facilitates formation of the composite material at the latest possible instance before the sample is vitrified. The reduction in the amount of time elapsed between composite sample formation and subsequent vitrification facilitates the capture of the target specimen(s) in non-equilibrium position(s). Moreover, it is possible to minimize, if not all together avoid, the target specimen(s) from returning to its/their preferred orientation(s), dissociation, aggregation, and/or adverse interaction(s) between the target specimen(s) and the air-water interface.

[0011] In an embodiment, the cryo-EM sample preparation device further comprises a first actuator configured to actuate the first applicator to dispense the first sample material and a second actuator configured to actuate the second applicator to dispense the second sample material. Having actuators that are configured to actuate the applicators to dispense the sample material onto the sample grid is advantageous because it facilitates automation of the sample preparation process. Moreover, the amount of sample dispensed onto the grid can be more precisely controlled, especially in comparison with other known techniques in which the sample material is manually applied (e.g., by pipetting) to the grid surface.

This means smaller amounts of sample material can be used from the outset, which is advantageous from a sample material conservation standpoint, and reduces the amount of wasted sample material that is removed from the grid prior to vitrification.

[0012] One or both of the first and second actuators may be provided in the form of solenoid, such as a solenoid piston. Solenoids are non-limiting examples of exemplary actuators that may be used in the cryo-

EM sample preparation device, however, it is to be understood that any alternative suitable to actuate the applicator(s), such as a pneumatic actuator, may be readily substituted into the device. Solenoid actuators, in particular, are desirable for their operability, and more particularly, because they can be configured to operate on a millisecond time scale. Further, solenoid actuators can be operated consistently and repeatedly, which in turn, ensures reproducibility.

[0013] A respective holder may be provided for each applicator in an embodiment of the cryo-EM sample preparation device. The first and second holders are arranged to secure the first and second applicators, respectively, in a first position and a second position. The holders may be custom-made and are not limited to any particular material or shape. By keeping the applicators secured in particular positions, sample preparation can be consistently reproduced from sample to sample. It is also beneficial to be able to have the applicator(s) in a set, constant location as this can assist with identifying if and where adjustment to the sample set-up may be needed.

[0014] The first and second positions are not particularly limited but are preferably oriented such that they each extend in a direction along a transverse axis (x) of the cryo-EM sample preparation device. In this regard, the first and second positions may be essentially perpendicular to a central longitudinal axis (Y) of the cryo-EM sample preparation device. This arrangement facilitates sample material application on the sample grid.

[0015] The first and second applicators may each comprise a microliter syringe in certain embodiments.

Microliter syringes are preferred because they are readily available, inexpensive, and the amount of sample material can be readily controlled. It is further preferable that these microliter syringes are gastight.

[0016] While the amount of sample material provided in one or both of the applicator(s) is not particularly limited, the first and second applicators are each adapted to accommodate a sample volume of 100 microliters or less in an exemplary embodiment. Such applicator(s) may, in some embodiments, contain a relatively small sample volume of, for example, 1-3 microliters.

[0017] In operation, the actuator is displaced a non-zero distance to effectuate actuation of an applicator to dispense sample material therefrom onto the sample grid. As such, it has been found that there is a correlation between the level of displacement of the actuator and the sample volume that is discharged from the applicator. In some embodiments, a displacement on the order of millimeters (mm) corresponds with a dispensed sample volume on the order of microliters (pL). In one exemplary non-limiting embodiment, a 2-3 mm displacement of an actuator piston corresponds with the dispensation of 2-3 pL of sample material onto the sample grid from a 100 microliter syringe.

[0018] In an embodiment, the first and second gas outlets are configured to emit a stream of gas. The stream(s) of gas emitted by the first and second gas outlets are preferably oriented such that they are essentially parallel to the sample grid. A stream of gas which is substantially parallel to the sample grid may act to create a gradient within the fluid across the grid surface, which in turn, can lead to the provision of a thickness of the sample material that is optimized for electron microscopy imaging. In other words, arranging the stream of gas such that it is essentially parallel to the sample grid is advantageous for facilitating removal of excess sample material therefrom.

[0019] The streams of gas emitted by each of the first and second outlets may be a laminar flow of gas.

Further, the flow of gas may be applied at a pressure in a range of 2 to 12 bar, preferably 4 to 10 bar, and more preferably in a range of 5 to 9 bar.

[0020] When a laminar flow of gas is emitted from one or both of the first and second gas outlets, the flow may be applied for a predetermined time period. This predetermined time period may be preferably 150 milliseconds or less, more preferably 100 milliseconds or less, and even more preferably between 50 and 100 milliseconds. Predetermined time periods that are out of range, either by being shorter or longer in duration, can generate layers of sample material that are either too thick, too thin, or even non-existent.

[0021] In an embodiment, the first and second gas outlets each comprise a pressurized gas nozzle. This is advantageous for enhancing control over the stream(s) of gas, in terms of both the directionality and the amount of gas emitted from the nozzles. The pressurized gas nozzles are not limited to any particular form.

However, in certain embodiments, it is preferred that the gas nozzles are in the form of flat air nozzles.

[0022] The stream of gas emitted from one or both of the first and second gas outlets may comprise compressed air or another generally inert gas which does not detrimentally interact with the sample material(s}). In one exemplary embodiment, the stream of gas comprises nitrogen gas.

[0023] The cryo-EM sample preparation device includes one or more guards to protect the cooling bath from having its contents disturbed, either by having its contents displaced therefrom or by allowing undesired material(s) to fall therein. In one embodiment, the sample preparation device comprises a single guard. In another non-limiting embodiment, the sample preparation device comprises two guards. However, it is to be understood that the number and/or arrangement of the guard(s) is/are not particularly limited provided that the chosen number and/or arrangement is suitable to protect the cooling bath from having its contents disturbed while the device is in the closed configuration.

[0024] In any case, when the sample preparation device is in the closed configuration, the guard(s) is/are arranged to shield the cooling bath from a stream of gas emitted from one or both of the first and second gas outlets. It may also be the case that the guard(s) prevent displaced sample material from falling down into the cooling bath prior to, during, or following application to the sample grid.

[0025] Regardless of the number of guards provided, the guard(s) is/are preferably arranged to be translatable along a transverse axis (xX) of the cryo-EM sample preparation device. This arrangement enables the guard(s) to move between the closed configuration, which shields the cooling bath from the stream(s) of gas emitted from one or both of the gas cutlets, and the open configuration, which permits the 5 sample grid to be plunged into the cooling bath.

[0026] In an embodiment, the sample grid is downwardly translatable along a central longitudinal axis (Y) of the cryo-EM sample preparation device and into the cooling bath.

[0027] It is preferred for the cooling bath to be a temperature controlled bath, as this may contribute to ensuring that the sample material on the sample grid is vitrified. The temperature(s) or range(s) of temperatures at which the cooling bath is controlled may be varied in accordance with one or more of the cooling fluid(s), sample material(s), and target specimen(s), as these factors may impact the temperature at which the sample becomes vitrified. In one non-limiting example, the cooling bath may be controlled at a temperature of -110°C or below. In other embodiments, the cooling bath may be controlled at a lower temperature or range(s) of temperatures still, such as at minus 150 degrees Celsius and below. And, in another non-limiting embodiment, the cooling bath has a preferred temperature of minus 180 degrees

Celsius or colder.

[0028] The cooling bath comprises a cooling fluid that is suitable to vitrify the sample material(s). In a non-limiting embodiment, it is preferred that the cooling fluid is capable of achieving a cryogenic temperature, e.g. negative 150 degrees Celsius or lower (the term ‘cryogenic’ generally being understood as referring to temperatures of minus 150 degrees Celsius and below). In another non-limiting embodiment, the cooling fluid may be capable of achieving a temperature of negative 180°C or colder. Likewise, it is also contemplated that the cooling fluid may be controlled at a temperature or range(s) of temperatures above -150°C, e.g., at minus 110°C and below. The cooling fluid is preferably selected from one of: liquid ethane, liquid ethane-propane, or liquid nitrogen.

[0029] The sample grid comprises a grid surface comprising a plurality of discrete zones and a porous film suspended there across. The plurality of discrete zones and the porous film are adapted to receive an amount of sample material therein. In one non-limiting example, the plurality of discrete zones are square in shape and spaced a distance (e.g., tens of microns) apart from each other. However, it is to be understood that the shape and sizing of the discrete zones is not particularly limited, and it is contemplated that the plurality of grid zones can take the form of any suitable shape and/or dimension(s). As for the porous film, one non-limiting example of a material from which the film may be formed is holey carbon.

Further, the sample grid may comprise a metal.

[0030] The cryo-EM sample preparation device is provided with a control unit. This enables aspects of the sample preparation process to be automated, which in turn, is advantageous for producing prepared samples that are consistently reproducible. Automation is also beneficial because it allows for precise timing of process steps, which is particularly advantageous for sample prepared on a millisecond time scale. The control unit may be configured in an embodiment to control the first and second applicators to respectively dispense the first and second materials therefrom onto a grid surface of the sample grid. Additionally or alternatively, the control unit may be configured to control one or both of the first and second gas outlets to deliver a stream of gas towards or directly onto a grid surface of the sample grid to remove excess sample therefrom.

[0031] Additionally or alternatively, the control unit may further be configured to actuate the guard(s) to move from the closed configuration to the open configuration. At the same time, the control unit controls the sample support to translate the sample grid into the cooling bath.

[0032] The cryo-EM sample preparation device further comprises an image capturing apparatus. The provision of an image capturing apparatus makes it possible to monitor and record each step of the sample preparation process in real-time. A preliminary assessment of the quality of the prepared sample(s) can then be performed based on the monitoring/recording of the sample preparation process. Poor quality samples, such as those which have poor distribution of the sample material(s) on the grid or those which carry little to no sample material at all can thus be readily identified and discarded instead of being saved for subsequent imaging by electron microscopy. The preliminary assessment can also help to inform whether the sample material(s) is/are actually present on the sample grid, whether the controlled stream of gas is distributing and/or removing excess sample material from the grid surface, and/or identify where adjustments are needed in the sample preparation set-up to remediate and/or obviate the occurrence similar defects in subsequent samples. Accordingly, both experimental resources and time can be saved.

[0033] The image capturing apparatus may, in certain embodiments, be a high-speed camera preferably operable to capture images at a rate of at least 500 frames per second with a video graphics arrays (VGA) resolution of 2 milliseconds per frame.

[0034] In an embodiment, the cryo-EM sample preparation device further comprises a light source. The light source may be included in the cryo-EM sample preparation device to assist in illuminating the sample grid to facilitate image capturing of the process by an image capturing apparatus.

[0035] How and when the first and second sample materials are deposited onto the sample grid is not particularly limited. It is contemplated that the application of the first and second sample materials may be varied in view of the sample material(s) being tested. In one non-limiting embodiment, the first and second sample materials are consecutively dispensed one after the other onto a grid surface of the sample grid. In another non-limiting embodiment, the first and second sample materials are simultaneously dispensed onto a grid surface of the sample grid.

[0036] In an embodiment, one of the first and second sample materials comprises a target specimen.

The target specimen is not particularly limited and can be readily tailored as desired between experiments.

In one non-limiting example, the target specimen may be one or more macromolecules. Other non-limiting exemplary target specimens may include any one or combination of: one or more viruses, one or more liposomes, one or more microbeads, one or more cells, DNA molecules, RNA molecules, one or more nucleotides, one or more lipids, and any metabolite or small molecule.

[0037] In an embodiment, one of the first and second sample materials comprises a macromolecule solution and the other sample material comprises a small molecule solution. The macromolecule and small molecule solutions are not particularly limited. Some non-limiting, exemplary solutions may include one or more of: peptides, nucleotides, substrates, inhibitors, and other compounds.

[0038] In a further aspect of the present disclosure, a sample for cryogenic electron microscopy is prepared according to the following method steps: providing a cryogenic electron microscopy sample preparation device as described herein; applying a first sample material and a second sample material on a grid surface of the sample grid, wherein the first and second sample materials mix to form a composite sample material; delivering a controlled stream of gas from one or both of the first and second gas outlets towards or directly onto the grid surface to remove excess sample material therefrom; submerging the sample grid in the cooling bath to vitrify the composite sample material in ice.

[0039] Preparation of a sample for cryogenic electron microscopy also includes capturing consecutive images of at least each of the steps of: applying the first sample material and the second sample material on the grid surface of the sample grid, wherein the first and second sample materials mix to form a composite material; delivering a controlled stream of gas from one or both of the first and second gas outlets towards or directly onto the grid surface to remove excess sample material therefrom; and submerging the sample grid in the cooling bath to vitrify the composite sample material in ice. Such consecutive images are preferably captured with a high-speed camera.

[0040] The first and second sample materials can be consecutively dispensed one after the other onto the grid surface. Alternatively, the first and second sample materials may be simultaneously dispensed onto the grid surface. In any case, the order and timing of application of the material(s} to the sample grid may be tailored to the specific sample material(s) being tested.

[0041] Dispensation ofthe first and second sample materials can be effectuated according to a particular dispensation scheme. The dispensation scheme is not particularly limited and may be tailored in respect of one or more parameters (e.g., target specimen(s), sample material(s), etc.) of a given experiment.

[0042] Similarly, delivery of the controlled stream of gas to the grid surface can be effectuated according to a particular gas delivery scheme and may also be specifically tailored according to one or more experiment-specific parameters. For example, a stream of gas from one or both of the first and second gas outlets may be delivered to the grid surface during a gas delivery period of 100 milliseconds or less. In another non-limiting embodiment, the gas delivery period lasts between 50 and 100 milliseconds. The gas delivery period may be shortened even further still in other embodiments to last between 25 and 50 milliseconds. Gas delivery periods within these ranges can be particularly advantageous for optimizing the thickness of the resultant layer(s) of sample material on the sample grid; time periods outside of these ranges, whether shorter or longer in duration, can form layer(s) of sample material that are either too thick or too thin for electron microscopy imaging.

[0043] Optionally, there may be a delay between application of the first and second sample materials on the grid surface and delivery of the controlled stream of gas onto the grid surface. Additionally or alternatively, there may be an optional delay between delivery of the controlled stream of gas onto the grid surface and submersion of the sample grid in the cooling bath. The timing(s) of the optional delay(s) is/are not particularly limited and may be specifically tailored to one or more experiment-specific parameters (e.g., target specimen(s), sample materials(s), etc.).

[0044] In one non-limiting example, the controlled stream of gas can be delivered with a delay of not more than 30 milliseconds, not more than 20 milliseconds, or not more than 10 milliseconds after the first and second sample materials are applied to the grid surface. It is also contemplated that in some embodiments, there may even be no delay between application of the sample materials to the sample grid and the subsequent delivery of the controlled stream of gas.

[0045] In another non-limiting example, the sample grid may be submerged in the cooling bath with a delay of not more than 25 milliseconds, not more than 15 milliseconds, or not more than 5 milliseconds after delivery of the controlled stream of gas to the grid surface. It is also contemplated that in some embodiments, there may be no delay at all between delivery of the controlled stream of gas and submersion of the sample grid into the cooling bath for vitrification.

Brief description of the drawings

[0046] The present invention will be discussed in more detail below, with reference to the attached drawings, in which:

[0047] Figure 1 shows an exemplary cryogenic electron microscopy sample preparation device.

[0048] Figure 2 shows an exemplary dual applicator holder.

[0049] Figure 3 shows an exemplary cryogenic electron microscopy sample preparation device in a closed configuration.

[0050] Figure 4 shows an exemplary cryogenic electron microscopy sample preparation device in an open configuration.

[0051] Figure 5 shows an exemplary control unit user interface.

[0052] Figure 6 shows a schematic illustration of an exemplary method of preparing a sample for cryogenic electron microscopy.

Description of embodiments

[0053] Figure 1 shows an embodiment of a cryo-EM sample preparation device (1) according to the present disclosure. The cryo-EM sample preparation device includes a sample support (10) configured to receive a sample grid (100). In certain embodiments, such as the non-limiting example shown in Fig. 1, the sample support may comprise means, such as tweezers or the like, for holding the sample grid in a preferred orientation. Here, the sample grid (100) is shown to be arranged along a central longitudinal axis (Y) of the cryo-EM sample preparation device, and in this regard, may be seen as generally oriented perpendicular or essentially perpendicular to a transverse axis (x) of the device.

[0054] The device includes dual applicators (21, 22), each of which is configured to comprise a sample material (201, 202). As can been seen in Fig. 1, the applicators are arranged to extend along the transverse axis (x) of the device, and in this particular embodiment, are also generally oriented perpendicular or essentially perpendicular to a grid surface of the sample grid onto which the sample material(s} may be applied. The applicators may be microliter syringes adapted to accommodate an exemplary sample volume of 100 microliters or less. It is further preferable that the applicators are gastight. It is also to be understood that the aforementioned microliter syringes are merely exemplary and that any known alternative(s) suitable for containing a sample material therein and applying it therefrom, may be readily substituted. Likewise, a sample volume of 100 microliters or less is non-limiting, and it is contemplated that the skilled person may select any size suitable for the desired cryo-EM sample to be prepared. The cryo-EM sample preparation device may also further comprise first and second holders (25, 26), such as those depicted in Figure 2.

These holders are arranged to secure the first and second applicators in respective first and second positions. This is advantageous because it allows for the sample material(s) to be applied at a precise position on the sample grid, not to mention inadvertent movement of the applicators can be avoided.

[0055] Turning back to Fig. 1, a respective actuator (23, 24) is also provided to actuate the first and second applicators to respectively dispense the first and second sample materials onto the sample grid. In the present example, the actuators comprise respective first and second solenoid actuators (231, 241).

However, it is to be understood that solenoid actuators are merely exemplary and any known alternative(s) suitable for actuating the applicators to dispense the sample material therein may be readily substituted in their place.

[0056] The cryo-EM sample preparation device has two gas outlets (31, 32), each configured to emit a stream of gas. As depicted in Fig. 1, the gas outlets are arranged on opposing sides of the sample support and, by extension, on opposing sides of the sample grid. The streams of gas are also preferably oriented parallel or essentially parallel to the sample grid. Arranging the gas outlets in this way allows the streams of gas emitted therefrom to be delivered to the opposing sides of the sample grid in a manner that is simultaneous, essentially symmetric, and/or so that the streams of gas are evenly distributed to the opposing surfaces of the sample grid. The emission of the streams of gas from the gas outlets also facilitates removal of excess sample from the sample grid. Consequently, the sample material remaining on the sample grid may take the form of a relatively thin layer of material having a thickness, for example, on the order of 100 nm or less. In one non-limiting example, such relatively thin layers of material, once vitrified, have been shown to demonstrate an average ice thickness in the range of 30 to 80 nm. This is particularly desirable for cryo-EM imaging and for optimizing high resolution structure determination of one or more target specimens in the sample material.

[0057] The cryo-EM sample preparation device also includes a cooling bath (50). The cooling bath is positioned along the central longitudinal axis (Y} and is located beneath the sample grid. By way of this arrangement, the sample grid can be translated downwardly and into the cooling bath to vitrify the sample material thereon with relative ease.

[0058] There is also at least one guard (40) provided at a position on the central longitudinal axis (Y) that is between the sample grid and the cooling bath. Fig. 1 depicts a non-limiting example of this aspect which includes two guards (40a, 40b). The one or more guards are arranged to extend in a transverse direction along a transverse axis (x) of the device. Each guard is translatable along the transverse axis between a closed configuration and an open configuration. In the closed configuration, the one or more guards are arranged to shield the cooling bath before and during sample material application to the sample grid. The guard(s) can prevent stray or excess sample material from entering the cooling bath. The guard(s) can additionally or alternatively shield the cooling bath from the stream(s) of gas emitted from the gas outlets and/or an excess sample material removed from the sample grid thereby. Figs. 1 and 3 each depict a non- limiting example of two guards arranged in the closed configuration, i.e. before and during sample material application.

[0059] Figure 4, by contrast, shows the two guards of Figure 3 in an open configuration. In this non- limiting example, the two guards have been translated along the transverse axis of the device in opposing directions away from each other. This opens up space along the central longitudinal axis (Y) for the sample grid to be translated downwardly and into the cooling bath below.

[0060] Turning back again to Fig. 1, the cryo-EM sample preparation device is equipped with an image capturing apparatus (70). The image capturing apparatus is configured to monitor and record the sample grid during sample preparation (i.e., including, but not limited to, prior to, during, and after sample application; removal of excess sample material from the sample grid; and subsequent disposition of the sample grid into the cooling bath for vitrification). The image capturing apparatus may, in certain embodiments, be provided in the form of a high-speed camera. The high-speed camera is preferably operable to capture images at a rate of at least 500 frames per second. It is further preferable that the image capturing apparatus have a video graphics array (VGA) resolution of 2 milliseconds per frame. This allows for each step of the sample preparation process, despite occurring on the millisecond time scale, to be monitored in real-time.

[0061] The cryo-EM sample preparation device may also have a light source (80). Providing a light source may assist with illumination of the sample preparation field, which by extension, may enhance image capture by the image capturing apparatus. The light source may be provided in the form of a torch/flashlight.

Such a torch (LED Lenser® P3) is shown in the sample preparation device of Figs. 3 and 4. However, it is to be understood that the aforementioned torch is merely exemplary and any known alternative(s) suitable for illuminating the sample preparation field may be readily substituted in its place.

[0062] In order to facilitate reproducible and consistent sample preparation, especially on a millisecond time scale, a control unit (60) is provided to automate aspects of the sample preparation device. This allows for precise timing, in terms of when each step (e.q., application of sample material(s) to the grid, removal of excess sample, vitrification) is performed during the sample preparation process and over a particular duration.

[0063] The control unit may be an electronic device, such as a laptop, a computer, or the like, with software loaded thereon and containing instructions for controlling one or more components of the cryo-

EM sample preparation device. In particular, the control unit may be configured to control one or more of: actuation of the applicators to dispense the respective sample materials therein onto the sample grid; delivery of a stream of gas towards or onto a grid surface of the sample grid from one or both of the gas outlets; simultaneous actuation of the guard(s) to move from the closed configuration to the open configuration and translation of the sample grid into the cooling bath; and operation of the image capturing apparatus.

[0064] An exemplary user interface for the control unit is shown in Figure 5. In this non-limiting example, the user interface has a number of different horizontal bars, each representing a particular aspect or step of the sample preparation process. The first horizonal bar at the top of the display controls the number of frames captured by the image capturing apparatus. Moving downward from the first horizonal bar, the next two bars control the timing of the application of the sample materials to the sample grid from the first and second applicators. In this particular non-limiting example, samples A and B are dispensed consecutively, one after the other, with sample A shown as being dispensed in a period of just 3 milliseconds (between 25 and 28 milliseconds) and sample B shown as being dispensed thereafter in a period of only 4 milliseconds (between 52 and 56 milliseconds). Continuing downward, the next horizontal bar controls the timing and duration of the delivery of the stream of gas to the sample grid from one or both of the gas outlets. In this example, the sample grid is dosed with a stream of gas for a 70 milliseconds, beginning at 77 milliseconds into the sample preparation process and ending at 147 milliseconds. An optional pause or delay between application of the sample materials to the sample grid and the emission of the stream of gas is also observable in this example. The delay between the end of the application of sample B and the start of the delivery of the stream of gas is about 20 milliseconds in duration (sample B ending at 56 ms followed by puffer start at 77 ms), however, it is to be understood that this time period is merely exemplary, and other durations or even no delay at all may be alternatively applied. Finally, at the bottom of the user interface, the last horizontal bar controls the timing of the translation of the sample grid into the cooling bath for vitrification, sometimes also referred to as ‘the plunge’. In this non-limiting example, there is a delay of less than 10 ms between completion of the delivery of the stream of gas and translation of the sample grid into the cooling bath. Again, this duration is merely exemplary, and other durations or even no delay at all may be alternatively applied.

[0065] Another aspect of the present disclosure, schematically illustrated in Figure 6, is a method of preparing a sample for cryogenic electron microscopy. In particular, a sample for cryo-EM can be prepared by: providing a cryogenic electron microscopy sample preparation as described herein (not shown); (b) applying a first sample material and a second sample material on a grid surface of the sample grid, the first and second sample materials mixing to form a composite material; (c) delivering a controlled stream of gas from one or both of the first and second gas outlets towards or directly onto the grid surface to remove excess sample material therefrom; and (d) submerging the sample grid in the cooling bath to vitrify the sample material in ice.

[0066] The order and timing of the application of the subject sample materials to the sample grid is not particularly limited. The first and second sample materials can be consecutively dispensed one after the other onto the grid surface, or they may be simultaneously dispensed onto the grid surface. In any case, application of the material(s) can be tailored to the specific sample material(s) being tested.

[0067] There may also be one or more optional delays between method steps. In particular, there may be a delay between application of the first and second sample materials on the grid surface and delivery of the controlled stream of gas onto the grid surface. Additionally or alternatively, there may be a delay between delivery of the controlled stream of gas onto the grid surface and submersion of the sample grid in the cooling bath. In any case, the timing(s) of the optional delay(s) is/are not particularly limited and may be specifically tailored to one or more experiment-specific parameters (e.g., target specimen(s), sample materials(s}, etc.).

[0068] Additionally, the method of preparing a sample for cryo-EM comprises capturing consecutive images of process steps, and in particular at least each of the steps of: applying the first sample material and the second sample material on the grid surface of the sample grid; delivering a controlled stream of gas from one or both of the first and second gas outlets towards or directly onto the grid surface to remove excess sample material therefrom; and submerging the sample grid in the cooling bath to vitrify the composite sample material in ice. Such consecutive images are preferably captured with a high-speed camera.

[0069] While the invention has been described herein by reference to certain embodiments, it is to be understood that modifications in addition to those described herein may be made to the structures and the techniques described herein without departing from the spirit and scope of the invention. Accordingly,

although specific embodiments have been described, they are examples only and are not limiting upon the scope of the invention.

Claims

Conclusions

Apparatus (1) for preparing a sample for performing cryogenic electron microscopy (cryo-EM), comprising: a sample support (10) configured to receive a sample grid (100), a first applicator (21 ) configured to include a first sample material (201), a second applicator (22) configured to include a second sample material (202), a first gas outlet (31), a second gas outlet (32), at least one guard (40) configured to be moved between a closed configuration and an open configuration, a cooling bath (50), a control unit (60), and an image capture device (70).

A device (1) for preparing a sample for cryo-EM, according to claim 1, further comprising a first actuator (23) configured to activate the first applicator (21) to deliver the first sample material (201). and also a second actuator (24) configured to activate the second applicator (22) to dispense the second sample material (202), and wherein the first actuator preferably includes a first solenoid (231), and the second actuator preferably comprises a second solenoid (241).

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, further comprising a first holder (25) for the first applicator (21), and a second holder (26) for the second applicator (22), wherein the first and second holders (25, 26) are provided to hold the first and second applicators (21, 22) in a first position and in a second position, respectively,

wherein the first and second positions preferably extend in a direction along a transverse axis (X) of the cryo-EM sample preparation device (1).

A device (1) for preparing a sample for cryo-EM, according to claim 3, wherein the first and second positions are substantially perpendicular to a central longitudinal axis (Y) of the device (1) for preparing a sample for cryo-EM. Preparing the sample for cryo-EM.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the first applicator (21) and the second applicator (22) each comprise a microliter syringe that is preferably designed to be gas-tight .

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein each of the first and second applicators (21, 22) is adapted to accommodate a sample volume of at most 100 microliter.

A device {1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the first and second gas outlets (31, 32) are each configured to deliver a gas flow that is preferably substantially parallel oriented to the sample grid (100) and provided to remove excess sample material from the sample grid (100).

Device (1) for preparing a sample for cryo-EM, according to claim 7, wherein the gas flow is a laminar gas flow under a pressure ranging from 2 bar to 12 bar, which is preferably is located in the range from 4 bar to 10 bar, and is preferably located in the range from 5 bar to 9 bar.

Device (1) for preparing a sample for a cryo-EM, according to claim 7 or claim 8, wherein the gas flow is supplied for a predetermined period of time, the length of which is preferably at most 150 ms, the length of which at is preferably at most 100 ms, and the length is preferably at most between 50 ms and 100 ms.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the first and second gas outlets (31, 32) each comprise a manifold for a pressurized gas, this manifold preferably is a flat air manifold.

A device (1) for preparing a sample for a cryo-EM, according to any one of the preceding claims, wherein a gas flow delivered from one or both of the first and second gas outlets (31, 32) comprises nitrogen gas.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, comprising two guards (40a, 40b) configured to be moved between the closed configuration and the open configuration.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the at least one protection (40) is provided to shield the cooling bath (50) from a gas flow that is delivered through one or both of the first and second gas outlets (31, 32) in the closed configuration.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the at least one shield (40) can undergo a translational movement along a transverse axis (x) of the preparatory device (1) for the cryo-EM sample, from the closed configuration to the open configuration.

Device {1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the sample grid (100) is capable of downward translational movement along a central longitudinal axis (Y) of the device (1) for cryo-EM preparing a sample for a cryo-EM, in the cooling bath (50).

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the cooling bath (50) is a temperature-controlled bath, preferably with a temperature of at most -110° C, better with a temperature not exceeding -150 °C, and even better with a temperature not exceeding -180 °C.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the cooling bath (50) is a liquid ethane bath, a liquid ethane-propane bath, or a liquid nitrogen.

Device {1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the sample grid (100) comprises a grid surface comprising a plurality of discrete zones and a porous foil, preferably carbon with holes , suspended over it, the plurality of discrete zones and the porous foil being adapted to accommodate a quantity of sample between them.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the sample grid (100) comprises a metal.

A device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the control unit (60) is configured to control the first and second applicators (21, 22) to respectively the first and second materials (201, 202} onto a grid surface of the sample grid (100).

A device (1) for preparing a sample for cryo-EM, according to any one of claims 1 to 20, wherein the first and second sample materials (201, 202) are delivered one after the other onto a grid surface of the sample grid (100).

A device (1) for preparing a sample for cryo-EM, according to any one of claims 1 to 20, wherein the first and second sample materials (201, 202) are simultaneously delivered to a grid surface of the sample grid (100 ).

A device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the control unit (60) is configured to connect one or both of the first and second gas outlets (31, 32) to deliver a gas flow toward or directly onto a grid surface of the sample grid (100) to remove excess sample therefrom.

A device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein the control unit (60) is configured to simultaneously activate the at least one protection (40) in order to to move from the closed configuration to the open configuration, and to arrange the sample grid (100) in the cooling bath (50) using a translational movement.

A device (1) for preparing a sample for cryo-EM, according to claim 1, wherein the image recording device (70) comprises a high-speed camera preferably operable to record images at a speed of at least 500 frames per second, with a video graphics array (VGA) resolution of 2 ms per frame.

Device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, further comprising a light source (80).

A device (1) for preparing a sample for a cryo-EM, according to any one of the preceding claims, wherein one of the first and second sample materials (201, 202) comprises a target specimen, preferably comprising one or more of: at least one macromolecule, at least one virus, at least one liposome, at least one microbead, and at least one cell.

A device (1) for preparing a sample for cryo-EM, according to any one of the preceding claims, wherein one of the first and second sample materials (201, 202) comprises a macromolecule solution, while the other sample material (201, 202) 202) includes a small molecule solution.

29. Method of preparing a sample for cryogenic electron microscope (cryo-EM), comprising:

a. providing a device for preparing a sample for a cryo-EM, according to any one of claims 1 to 28;

b. applying a first sample material and a second sample material to a grid surface of the sample grid, wherein the first and second sample materials mix to form a composite sample material;

c. delivering a controlled gas flow from one or both of the first and second gas outlets toward or directly onto the grid surface to remove excess sample material therefrom;

d. immersing the sample grid in the cooling bath to vitrify the composite sample material into ice; and e. recording consecutive images of at least each of steps (b) to (d).

The method of claim 29, wherein the first and second sample materials are sequentially delivered to the grid surface.

The method of claim 29, wherein the first and second sample materials are simultaneously delivered to the grid surface.

A method according to any one of claims 29 to 31, wherein the controlled gas flow is delivered with a delay of not more than 30 ms following step (b), preferably with a delay of not more than 20 ms following step ( b), even better with a delay of no more than 10 ms following step {b}, and preferably without delay following step (b).

A method according to any one of claims 29 to 32, wherein the sample grid is immersed in the cooling bath with a delay of not more than 25 ms following step (c), preferably with a delay of not more than 15 ms following step (c), even better with a delay of no more than 5 ms following step (c), and preferably without delay following step (c).