Title

GRIPPER UNIT AND TRANSFER SYSTEM

Technical Field

[0001 ] The present invention relates to a gripper unit for gripping and transferring support structures, in particular electron microscopy grids, from a temperature-controlled stage into a liquid cryogen containing vessel and to a respective transfer system.

Background Art

[0002] For the automation of sample preparation for cryo-electron microscopy (cryo- EM) remotely controlled grippers for manipulating small objects of less than 10 mg weight, like for example electron microscopy grids (EM grids), are used. Typical aqueous protein sample layers are thinner than 1 pm to warrant acquisition of atomic scale resolution projections of the proteins. Such films are unstable because they evaporate extremely fast during transport of the grid from sample deposition into the liquid cryogen, which is required for vitrification. The transportation of such EM grids is generally critical and has to be carried out under specific conditions in order to avoid unwanted respectively uncontrolled evaporation of the sample layers since films of varying thickness and quality usually lead to a reduction in image quality or salt artifacts.

[0003] In WO 2018/073242 A1 a method for preparing a sample is described, wherein the sample is provided as a thin film on a support structure and the temperature of the support structure is adjusted to a value above the dew point temperature of the environment to decrease the film thickness. Further, light is directed at the support structure, an intensity value of the transmitted light is measured, and the support structure is automatically inserted into a liquid cryogen dependent on the measured intensity value. The application further relates to a system comprising a support structure, a temperature-controlled stage for keeping the support structure at a pre-defined temperature, a transfer mechanism for moving the support structure into a container containing a liquid cryogen, a light source, a photodetector for measuring an intensity value of the transmitted light, and a control device for triggering the transfer mechanism dependent on the measured intensity value. The combination of a temperature-controlled stage and an optical measurement device consisting of the light source and the photodetector advantageously allows controlled sample evaporation and preparation of cryo-grids at a defined and reproducible film thickness. This improves image quality of electron micrographs and avoids salt artifacts by uncontrolled water evaporation. This technique has proven to be effective in practice; however, there are some deficiencies remaining particularly during transfer of the support structures from the temperature-controlled stage to the container with the liquid cryogen (e.g., liquid ethane).

[0004] Therefore, there is still a need for further improved components for ensuring also an optimum transfer operation for the support structures, i.e. in particular from a temperature-controlled stage to a liquid cryogen container.

[0005] Also, there is a need to provide means for synchronous activation of the sample by rapid mixing, by illumination of photolabile caged compounds, or by spraying activating molecules onto the thin sample layer a few ms before insertion in the liquid cryogen.

[0006] Finally, there is a need to passivate the air-water interface by spraying effector molecules onto the thin protein solution layer.

Disclosure of the Invention.

[0007] According to the invention these needs are settled by a gripper unit as it is defined by the features of independent claim 1 and by a transfer system as it is defined by the features of independent claim 16. Preferred embodiments are subject of the dependent claims.

[0008] In one aspect, the invention relates to a gripper unit for gripping and transferring support structures, in particular electron microscopy grids. The gripper unit includes gripper fingers being configured for gripping and releasing the support structure, wherein the gripper fingers being made of a heat conductive material. The gripper unit further includes a temperature adjusting element being configured for adjusting the temperature of the gripper fingers to a predetermined value. [0009] The “transfer” of the support structures respectively the electron microscopy grids (EM grids) generally includes transporting the EM grids from a temperature-controlled stage, which is usually arranged on a positioning unit in a horizontal plane to an opening of a container filled with liquid cryogen into which the EM grid is immersed respectively plunged in a vertical direction and with a certain speed. Such EM-grids usually have a diameter of about 3 mm and a thickness of about 25 pm and are usually made of copper. During the writing process they are clamped on a platform which is cooled to a temperature close to the dew point. This temperature is critical since the written aqueous layer is thinner than 1 pm and may evaporate quickly.

[0010] The “gripper fingers” can for example be in the form of a standard tweezer or in the form of two separate gripper fingers from which at least one is movable relative to the other. The separate gripper fingers may be formed in a customized manner from materials with good heat conductivity.

[0011 ] Preferably, the temperature adjusting element is configured to adjust the temperature of the gripper fingers close to a dew point temperature when the gripper fingers are in contact with the support structure. Close to the dew point temperature is defined herein as including a range of about -2° C to + 20 °C, 0° C to 10° C and 0° C to 5° C. In this manner, unwanted respectively uncontrolled sample evaporation can be prevented and a particularly defined and reproducible film thickness of the EM grids can be achieved.

[0012] The “dew point” is understood to be the temperature to which air must be cooled to become saturated with water vapor, assuming constant air pressure and water content. When cooled below the dew point, moisture capacity is reduced and airborne water vapor will condense to form liquid water known as dew. When this occurs via contact with a colder surface, dew will form on that surface. The dew point is affected by humidity. When there is more moisture in the air, the dew point is higher. When the temperature is below the freezing point of water, the dew point is called the frost point, as frost is formed via deposition rather than condensation.

[0013] Preferably, the temperature adjusting element is configured to adjust the temperature of the gripper fingers such that the gripper fingers are defrosted when the gripper fingers are not in contact with the support structure. The term “defrosting” is defined herein as including a temperature of about 15 °C above the dew point, preferably of about 10 °C above the dew point and even more preferred of about 5 °C above the dew point. In this manner, a new EM grid to be transferred is not impaired by moist gripper fingers, which improves the overall efficiency of the gripper unit.

[0014] Preferably, the temperature adjusting element is in the form of a Peltier element. Peltier elements have a relatively small size and a relatively low weight and do not require any movable components or gases or liquids. However, the application of alternative temperature adjustment is conceivable.

[0015] Preferably, the gripper is dried by a flow of dry air to ensure that the gripper is not wet when a new EM grid is to be gripped.

[0016] Preferably, the gripper unit further includes a shielding compartment into which the gripper fingers are retractable, wherein in particular the shielding compartment is in the form of a cover surrounding the gripper fingers and the EM grid in their retracted state. In this manner a controlled climate (i.e. , in particular an actively controlled humidity) can be ensured during transfer operations. Thereby, the shielding compartment is preferably configured to be kept at a predetermined relative humidity and/or at a predetermined temperature close to the temperature of the gripper fingers. The shielding compartment also serves as a protection against „wind“, i.e., it will prevent a sample from drying out.

[0017] Preferably, the gripper unit includes a nozzle device being configured to spray the activating molecule onto the thin protein sample layer a few ms before insertion in the liquid cryogen.

[0018] Preferably, the gripper unit includes an ultrasonic activator being configured to promote rapid mixing of two separate sample layers a few ms before insertion in the liquid cryogen.

[0019] Preferably, the gripper unit includes an activation light source being configured to illuminate photolabile caged activators in the sample layer a few ms before insertion in the liquid cryogen.

[0020] Preferably, the gripper unit includes a further nozzle device being configured for injecting aerosols into the shielding compartment, the aerosols preferably carrying surface-active substances. In this manner it is possible to transport active solutes to a sample surface on an EM grid prior to vitrification to manipulate an air-water interface of the sample or to activate the sample.

[0021 ] Preferably, the gripper fingers comprise a first gripper finger and a second gripper finger being configured to be movable relative to each other in a vertical and/or horizontal direction. Thereby, in particular, a motor as preferred drive means may drive the upper gripper finger up and down relative to the lower gripper finger which is generally stationary. The motor may comprise a pneumatic, an electric or a mechanical drive mechanism. It is however also conceivable that either both gripper fingers may be driven by the motor or only the lower gripper finger may be driven by the motor. The gripper fingers may be designed individually in accordance with specific customer needs.

[0022] However, for most applications it is also a preferred embodiment when the gripper fingers are in the form of a (standard) tweezer. Such solution is particularly cost efficient. In such case the upper gripper finger of the tweezer is usually actuated by means of a pusher which is driven by a drive means respectively motor as described above.

[0023] Preferably, in the shielding compartment a temperature sensor and a relative humidity sensor are arranged for monitoring a temperature and a relative humidity within the shielding compartment. In this manner a particularly efficient climate control can be ensured.

[0024] Preferably, the gripper unit further includes an inertial measurement unit (IMU) for monitoring movements and accelerations of the gripper unit. Thereby, in particular, a six degrees of freedom IMU (6 DOF IMU) is placed on a holding means for the movable gripper finger or for the pusher (i.e. in case of a tweezer). By means of the IMU, the transfer movements and the plunging movements of the gripper unit may be monitored and controlled in a particularly precise manner.

[0025] In another aspect, the invention relates to a transfer system for transferring a support structure, in particular an electron microscopy grid, from a substantially horizontal position in a substantially vertical position. The transfer system comprises a gripper unit as described hereinbefore, a drive unit being operatively connected to the gripper unit for performing translational movements of the gripper unit and a rotation unit being operatively connected to the gripper unit for performing rotational movements of the gripper unit.

[0026] Preferably, the drive unit comprises a mechanical, an electronic and/or a pneumatic drive mechanism. Likewise, the rotation unit preferably comprises a mechanical, an electronic and/or a pneumatic drive mechanism.

[0027] Preferably, the pneumatic transfer mechanism further includes a plunger motor being operatively connected to the rotation unit for performing translational movements of the rotation unit together with the gripper unit and the drive unit. Advantageously, the plunger motor comprises a high speed controllable proportional valve. In this manner the speed respectively acceleration of the gripper unit may be controlled in a very precise and effective manner.

[0028] During the transfer process, the rotation unit at first moves the gripper unit downwards wherein the EM grid is retracted in the shielding compartment. A permanent magnet usually serves for establishing a connection with a vertical rail. This ensures that the gripper unit may then be accelerated by means of the plunger motor to a speed of about 1 m/s whereby a first (vertical) extension is achieved via the plunger motor rod. Before the EM grid hits the liquid ethane in the respective container the drive unit accelerates in a second acceleration step the gripper unit to a plunging speed of about 2 m/s whereby a second (vertical) extension is achieved via the pushrod of the drive unit. Usually, the second extension is smaller than the first extension. This process is monitored by the IMU and controlled by a respective control unit.

Brief Description of the Drawings

[0029] The method according to the invention are described in more detail hereinbelow by way of an exemplary embodiment of the present invention and with reference to the attached drawings, in which:

Fig. 1 shows a schematic side view of an inventive gripper unit in an inventive transfer system with extended gripper fingers in a basic position. Fig. 2 shows a schematic view of the opposite side of the inventive gripper unit in an inventive transfer system with extended gripper fingers according to Fig. 1 ;

Fig. 3 shows a schematic side view of the inventive gripper unit in an inventive transfer system with retracted gripper fingers moving from the basic position via an intermediate position into an abutment position;

Fig. 4 shows a schematic side view of the other side of the inventive gripper unit in an inventive transfer system with extended gripper fingers according to Fig. 2 in the end position;

Fig. 5 shows a schematic side view of an embodiment of the inventive gripper unit with two separate gripper fingers;

Fig. 6 shows a schematic side view of an embodiment of the inventive gripper unit with two gripper fingers in the form of a tweezer with the gripper fingers in the closed state; and

Fig. 7 shows a schematic side view of an embodiment of the inventive gripper unit open state.

Description of Embodiments

[0030] In the following description certain terms are used for reasons of convenience and are not intended to limit the invention. The terms “right”, “left”, “up”, “down”, “under" and “above" refer to directions in the figures. The terminology comprises the explicitly mentioned terms as well as their derivations and terms with a similar meaning. Also, spatially relative terms, such as "beneath", "below", "lower", "above", "upper", "proximal", "distal", and the like, may be used to describe one element's or feature's relationship to another element or feature as illustrated in the figures. These spatially relative terms are intended to encompass different positions and orientations of the devices in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be "above" or "over" the other elements or features. Thus, the exemplary term "below" can encompass both positions and orientations of above and below. The devices may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein interpreted accordingly. Likewise, descriptions of movement along and around various axes include various special device positions and orientations.

[0031 ] To avoid repetition in the figures and the descriptions of the various aspects and illustrative embodiments, it should be understood that many features are common to many aspects and embodiments. Omission of an aspect from a description or figure does not imply that the aspect is missing from embodiments that incorporate that aspect. Instead, the aspect may have been omitted for clarity and to avoid prolix description. In this context, the following applies to the rest of this description: If, in order to clarify the drawings, a figure contains reference signs which are not explained in the directly associated part of the description, then it is referred to previous or following description sections. Further, for reason of lucidity, if in a drawing not all features of a part are provided with reference signs it is referred to other drawings showing the same part. Like numbers in two or more figures represent the same or similar elements.

[0032] Fig. 1 shows a schematic side view of an inventive gripper unit 1 in an inventive transfer system 20 with extended gripper fingers 10, in a horizontal position, e.g., for taking up an EM-grid. The gripper unit 1 includes the slide means 7 to which the other components of the gripper unit 1 are directly or indirectly mounted and which is operatively connected with a pushrod 15 of the pneumatic drive unit 8. The components which are directly or indirectly mounted at the slide means 7 of the gripper unit 1 include for instance the cover 13 forming the shielding compartment 2 into which the gripper fingers 10 retract the EM-grid, the IMU 4 for monitoring the transfer process, rh/t sensor 3 for monitoring relative humidity and temperature within the shielding compartment 2, chemicals supply 11 a and climate supply 11 b by means of which humid air and/or aerosols may be introduced via application nozzle 32 in the shielding compartment 2 for providing a controlled climate therein or for activating the sample in the thin aqueous layer, an ultrasound activator 21 for mixing two separate sample layers, an activation light source 29 for activating caged compounds and a gripper temperature sensor 26. The drive means for the gripper fingers 10 and the respective holding structures will be described further below.

[0033] The transfer system 20 generally comprises the gripper unit 1 , the rotation unit 9, the drive unit 8, which is usually in the form of a pneumatic motor and plunger motor 23 which is preferably also pneumatically driven. Here, the pushrod 15 is in the extended position. The transfer system 20 is connected to the rotation unit 9, which is operatively connected to the plunger motor 23. The rail 12 extending vertically downwards form the rotation unit 9 thereby serves as a stop means for the transfer system 20. The rail 12 may also comprise a rotation damper 24 and a permanent magnet 12a for ensuring an optimal holding function. The plunger motor 23 preferably comprises a high-speed controllable proportional valve and is operatively connected to the rotation unit 9, the drive unit 8 and the gripper unit 1 and serves for providing the pressurized air required for the pneumatic transfer movements respectively accelerations.

[0034] The transfer system further comprises water supply 11 c and pressurized air supply 11 d as well as electrical connection 25.

[0035] Fig. 2 depicts the opposite side of the gripper unit 1 of Fig. 1 with extended gripper fingers, i.e., in a position for taking up an EM-grid from e.g., a temperature- controlled stage (nor shown). Here, one can see extension damper 28 and further the Peltier element 6 for providing the necessary cooling/heating. Just above the gripper fingers one can see chemical application nozzle 31 . The gripper fingers with the EM-grid are retracted back into the shielding compartment 2 for the transfer process. Thereby, the rotation unit 9 is actuated to rotate around rotation axis R to bring the gripper unit 1 via an intermediate position of the gripper unit T into the vertical position of the gripper unit 1 ” and in abutment with rail 12 as illustrated in Fig. 3 The permanent magnet 12a in the rail 12 provides for a firm hold. In the vertical positon the gripper fingers are accelerated in a two-step acceleration operation to a speed of about 1 m/s first by the plunger motor 23, resulting in extension Ei of plunger motor rod 23a, and then, shortly before hitting the liquid ethane, to a plunging speed of about 2 m/s by the drive unit 8, resulting in extension E ₂ of pushrod 15, as shown in Fig. 4. Here, the end position of the gripper unit T” is reached in which the EM-grid is plunged into a cryogen liquid as for example liquid ethane. The transfer process is monitored by the IMU 4.

[0036] After vitrification of the EM-grid the gripper unit discharges the latter in a holder which is preferably cooled by liquid nitrogen. Then, the transfer system 20 is moved back in its starting position. The Peltier element 6 thereby heats respectively defrosts the gripper fingers 10 and the cover 13 in order to make the gripper unit ready for a new preparation cycle. Preferably, the gripper unit 1 is dried by a flow of warm air. The entire transfer system 20 and transfer process is controlled by means of control unit 30 which is directly or remotely connected to the system. [0037] Fig. 5 illustrates a schematic side view of an embodiment of the inventive gripper unit 1 having two separately mounted gripper fingers 10a and 10b from which the upper gripper finger 10a is driven by drive means 5 and the lower gripper finger 10b is stationary. The drive means 5 is usually in the form of a pneumatic motor which is provided with pressurized air by means of the pressurized air supply. The upper gripper finger is mounted at a holding means 16 which is moved in the vertical direction (i.e. up and down) by the pneumatic gripper drive means 5. The Peltier element 6 is operatively connected to the upper gripper finger 10a via the holding means 16 and the slide means 7 and to the lower gripper finger 10b via the slide means 7. The gripper fingers 10a, 10b have been brought in the extended positon by pushrod 15 and are slightly opened. In this position the gripper fingers 10a and 10b may grip an EM-grid. Connections 17a and 17b are for pressurized air (for rotation two connections are required).

[0038] In Figs. 6 and 7 schematic side views of an embodiment of the inventive gripper unit 1 with two gripper fingers 10a, 10b in the form of a standard tweezer are shown in the closed state (Fig. 6) and in the open state (Fig. 7). Here, the upper gripper finger 10a is manipulated by a pusher 16a which is arranged at the underside of the holding element 16 respectively ultrasonic activator 21. The holding element 16 is moved in the vertical direction up and down by the pneumatic motor 5. In order to open the gripper fingers, the pneumatic drive means 5 moves the holding element 16 in the upward direction to release the pusher 16a from the upper gripper finger 10a. In order to close the gripper fingers, the pneumatic drive means 5 moves the holding element 16 in the downward direction to exert pressure onto the upper gripper finger 10a by means of the pusher 16a. The humidity and temperature sensor 3 is mounted together with the IMU at the holding element 16. In the rear portion of the gripper unit one can see the pushrod 15 of the drive unit 8 attached at the rear end of slide means 7. Extension damper 28 is also visible.

[0039] The present disclosure also covers all further features shown in the Figs, individually although they may not have been described in the afore or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the invention or from disclosed subject matter. The disclosure comprises subject matter consisting of the features defined in the claims or the exemplary embodiments as well as subject matter comprising said features. [0040] Furthermore, in the claims the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single unit or step may fulfil the functions of several features recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. The terms “essentially”, “about”, “approximately” and the like in connection with an attribute or a value particularly also define exactly the attribute or exactly the value, respectively. The term “about” in the context of a given numerate value or range refers to a value or range that is, e.g., within 20%, within 10%, within 5%, or within 2% of the given value or range. Components described as coupled or connected may be electrically or mechanically directly coupled, or they may be indirectly coupled via one or more intermediate components. Any reference signs in the claims should not be construed as limiting the scope.

[0041 ] The control unit 30 includes a computer program that may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. In particular, e.g., a computer program can be a computer program product stored on a computer readable medium which computer program product can have computer executable program code adapted to be executed to implement a specific method such as the method according to the invention. Furthermore, a computer program can also be a data structure product or a signal for embodying a specific method such as the method according to the invention.

List of reference numbers:

1 gripper unit (starting position)

T gripper unit (intermediate position)

1 ” gripper unit (vertical position)

1 gripper unit (end position)

2 chamber

3 sensor (rh/t)

4 IMU motor (gripper drive means) Peltier element slide means drive unit (pneumatic) rotation unit gripper fingers a first gripper finger b second gripper finger a chemicals supply b climate supply c water d pressurized air supply rail a permanent magnet cover pushrod holding means a pusher a connection b connection transfer system ultrasound activator rotary base plunger motor a plunger motor rod rotation damper electrical connection gripper temperature sensor temperature sensor (heat sink) extension damper activation light control unit application nozzle (chemicals) application nozzle (aerosols) Ei first extension

E2 second extension

R rotation axis