Control system for liquid filling level and microscope stage including such system

Abstract

The inventive concept relates to a system (100) for controlling a filling level (X) of a liquid-filled reservoir (110), the system (100) comprising: a reservoir (110) configured to be filled with a liquid up to at least a maximum filling level (Xmax); a probe (120) including a probe body (122), at least a part of the probe body (122) extending in a height dimension of the reservoir (110) from a first position (126) to a second position (124), the first position (126) being lower than the second position (124), the second position (124) being higher than the maximum filling level (Xmax), and the probe (120) further including a temperature-dependent sensor (128) configured to generate a signal based on a temperature at the second position (124), the temperature depending on the filling level (X); and a controller (250) for controlling the filling level (X) of the reservoir (110) according to the sensor signal such that the filling level (X) remains constant.

Description

Technical Field

[0001] The inventive concept relates to a system for controlling the filling level of a liquid-filled reservoir, and a microscope stage of a microscope comprising such a system. More specifically, the system can be used to control the filling level of cryogenic liquids, in particular the filling level of cryogenic liquids in a reservoir of a microscope stage used in the field of cryomicroscopy. Background Art

[0002] Cryomicroscopes in the sense of the present application specifically include cryo-optical microscopes and cryo-electron microscopes. Samples to be examined with a cryomicroscope are mostly prepared in advance by a method called cryofixation. In this method, the aqueous sample is frozen very rapidly (cryofixed) to a temperature below -150 °C, i.e., the sample is cooled very rapidly to avoid the formation of ice crystals. It has been shown that cryofixation is particularly suitable for the study of structural biology. The object to be studied (such as cells, enzymes, viruses or lipid layers) is thus embedded in a thin vitrified ice layer. The great advantage of cryofixation is that biological structures can be obtained in their natural state. For example, biological processes can be stopped at any point in time by cryofixation and studied in this vitrified state, for example, in a cryo-electron microscope, or also in an optical microscope with corresponding sample cooling. In this context, cryo-optical microscopes are mainly used to locate relevant regions (regions of interest) of the sample, which can be noticed and then relocated and observed in more detail in a cryo-electron microscope.

[0003] In order not to affect the quality of the frozen samples, it is very important that these frozen samples are transferred between the processing units used (such as cryofixation devices, freeze-fracture devices and coating devices) and the analysis devices (in this case mainly cryo-optical microscopes and cryo-electron microscopes) in a cooled and contamination-free, in particular anhydrous, manner. A sample transfer device (such as the sample transfer device disclosed in US10,144,010B2) can be used to transfer samples between the processing unit and / or the analysis unit under cryogenic conditions.

[0004] As pointed out above, the samples to be examined need to be processed under cryogenic conditions at all times. Contamination or devitrification can significantly reduce the success rate of the process. To identify regions of interest before starting the time-consuming and expensive cryo-electron microscopy (cryo-EM) steps, such as cryo-scanning electron microscopy (cryoSEM), cryo-transmission electron microscopy (cryoTEM), or cryo-electron tomography (cryoET), cryo-optical microscopy is typically applied. By using cryo-optical microscopy, regions of interest in the nanometer range can be identified within the cell volume (millimeter range). The regions of interest are then retraced in cryo-EM, thus significantly accelerating the analysis process. For this purpose, the optical microscope, at least the optical microscope stage, must be used under cryogenic conditions. The sample (usually the sample on a sample carrier, also called a sample grid) is loaded into a sample transfer device, also called a cryo-correlative light electron microscopy (cryo-CLEM) shuttle, and then transferred to the cryo-stage of the optical electron microscope.

[0005] The cryo-stage of the optical microscope preferably includes a cryogenic liquid refill container connected to the cryo-stage, typically a Dewar container for liquid nitrogen. WO 2016 / 016000 A1 proposes using internal cooling of the components of the microscope stage by means of a tube through which a cryogenic liquid (liquid nitrogen) flow is conveyed. However, in order to avoid temperature variations, it is necessary to provide a constant cryogenic liquid flow. In practice, however, the cryogenic pump that conveys the cryogenic liquid from the refill Dewar container only conveys a variable amount of cryogenic liquid, especially at low flow rates. On the other hand, the temperature sensors used in such known cryo-stages very quickly detect temperature deviations. This results in problems with the corresponding feedback control and temperature fluctuation behavior.

[0006] In view of the above problems, there is a need to improve the temperature control of the samples on the cryo-stage of a cryo-microscope. Although certain applications in cryo-microscopy have been described above, other applications can also be envisaged, especially those using another liquid filled into a reservoir. Summary of the Invention

[0007] The object of the inventive concept is to provide a system for controlling the filling level of a reservoir filled with a filling liquid, especially a cryogenic liquid, which system is especially for use in the cryo-stage of a cryo-microscope, and using such a system minimizes the risk of contamination or devitrification and possible sample drift. Generally, the inventive concept can be used to control the filling level of reservoirs filled with other types of liquids in other applications.

[0008] The inventive concept provides a system for controlling the filling level of a liquid-filled reservoir according to claim 1. The system includes: the reservoir configured to be filled with liquid up to at least a maximum filling level; a probe including a probe body, at least a part of which extends from a first position to a second position in the height dimension of the reservoir, the first position being lower than the second position, and the second position being higher than the maximum filling level, and the probe further including a temperature-related sensor configured to generate a sensor signal based on the temperature at the second position, which depends on the filling level; the system further includes a controller for controlling the filling level of the reservoir according to the sensor signal such that the filling level remains constant.

[0009] In terms of the application of the cryostage discussed above, the system according to the inventive concept controls the cryogenic liquid level of the reservoir of the cryostage according to the sensor signal such that the filling level of the cryogenic liquid in the reservoir remains constant. Accordingly, a constant temperature at the second position is achieved, which in turn ensures a constant temperature at the sample position, which is typically above and close to the second position. Thus, the risks of devitrification, contamination, and possible sample drift are minimized.

[0010] The inventive concept can also be used in other applications where the temperature at a position above the maximum filling level of the liquid in the reservoir depends on the corresponding filling level of the liquid in the reservoir. By keeping the filling level constant, a constant temperature at this position can be achieved.

[0011] The temperature-related sensor can be in contact with the second position or can be a non-contact sensor to generate a signal according to the temperature at the second position. This temperature can be the ambient temperature of the second position, such as the temperature of the vaporized cryogenic liquid at the second position, or the temperature of the probe body at the second position, or a temperature determined by the temperature of the probe body and the ambient gas temperature at the second position, depending on the arrangement and type of the sensor.

[0012] It should be noted that the sensor signal can be calibrated such that the system can determine the filling level according to the detected sensor signal, or vice versa, so as to determine the temperature at the second position according to the filling level present in the reservoir. In the case where the cryostage of a microscope has a system according to the inventive concept, the higher the filling level of the cryogenic liquid, the lower the temperature at the sensor. The temperature-related sensor can be calibrated, for example, to be a predetermined number of degrees higher than the boiling point temperature (evaporation temperature) of the cryogenic liquid such that the controller can keep the level of the cryogenic liquid in the reservoir at a constant desired level.

[0013] Preferably, the relationship between the temperature-related sensor signal and the filling level is a linear relationship. Such a linear relationship can be achieved in a system according to the inventive concept by selecting a suitable material for the probe body, its arrangement in the reservoir, and by using a suitable temperature-related sensor, as will be discussed below.

[0014] In an embodiment, the first position on the probe body is located at or below the minimum filling level. Especially in the case of a thermally conductive probe body, when the filling level reaches the first position on the probe body, the temperature at the second position immediately drops. Therefore, in this case, it is sufficient to position the first position on the probe body at the minimum filling level.

[0015] In an embodiment, in the height dimension of the reservoir, the first position is laterally offset relative to the second position. In other words, the probe body extends diagonally through at least a part of the interior of the reservoir. In a projection on a horizontal plane (when viewed from above), the probe body has a length according to the lateral displacement. In a projection on a vertical plane, the length of the probe body is equal to or less than the length of the probe body itself. With this arrangement, the length of the probe body immersed in the liquid can be greater than the maximum filling level. This improves the accuracy of the sensor signal. In this context, preferably, the probe body extends through most of the reservoir in the width dimension of the reservoir, i.e., through more than 50% of the width of the reservoir. This increases the length of the probe body and thus increases the dynamic range and accuracy of the temperature-related signal, and thus also improves the accuracy of controlling the filling level constant.

[0016] In this embodiment, preferably, the angle of inclination of the probe body relative to the bottom side of the reservoir is equal to or less than 65°, 55°, 45°, 35° or 25°. With such an angle of inclination, a substantially diagonal arrangement of the probe body within the reservoir can be achieved. The minimum angle of inclination depends on the relationship between the maximum width dimension of the reservoir and the height of the reservoir, and the probe body extends diagonally from the bottom of the reservoir to the top of the reservoir on the other side.

[0017] In an embodiment, the probe body is in the form of a beam, rod, wire or sheet. The specific form usually also depends on the space requirements within the reservoir and the material used. Preferably, the probe body is made of metal, especially steel, more particularly X5CrNi18-10 (also known as 1.4301). The thermal conductivity of another material for the probe body should not exceed the thermal conductivity of this steel in order to be able to maintain the linear relationship between the temperature-related sensor signal and the filling level. Further, a greater dynamic range can be achieved by using a probe body material with a lower thermal conductivity.

[0018] In an embodiment, the probe body is reinforced in the portion located above the maximum filling level. This reinforcement has proven useful in view of the higher accuracy and greater dynamic range of the relationship between the temperature-related sensor signal and the filling level.

[0019] In an embodiment, the temperature-related sensor is mounted on the probe body at a second position of the probe body and specifically detects the temperature at the second position on the probe body. In this embodiment, it is useful to use a thermally conductive probe body having the above specifications. Further, it is preferable to use a resistance thermometer as the temperature-related sensor. A suitable resistance thermometer is, for example, a PT1000-sensor, which is used to measure the resistance value according to the temperature at the second position (its position). This temperature depends on the temperature of the probe body at the second position and the temperature of the surrounding gas at the second position.

[0020] In an embodiment, the system according to the inventive concept further includes: a supply line for supplying a liquid to the reservoir; a flow regulator for regulating the flow rate of the liquid entering the reservoir; and the controller connected to the temperature-related sensor and the flow regulator for controlling the filling level of the reservoir by adjusting the flow rate of the liquid entering the reservoir according to the sensor signal. The flow regulator may include an electric pump and / or a control valve. Such a flow regulator may be arranged in the supply line or between the liquid refill container (such as a Dewar container) and the supply line. The controller receives the temperature-related sensor signal and, in the case of a deviation from the set value, correspondingly controls the flow regulator to increase or decrease the flow rate of the liquid. Thus, by controlling the sensor signal to be equal to the set value, the filling level in the reservoir can be kept constant.

[0021] As described above, it is preferable that the liquid is a cryogenic liquid.

[0022] Further, the inventive concept also relates to a microscope stage, in particular a cryomicroscope stage, of a microscope, which includes the system according to the inventive concept as described above. All features related to the system according to the inventive concept as described above also apply to the microscope stage, in particular the cryo-stage, according to this other aspect of the inventive concept.

[0023] It should be noted that the above features of the embodiments of the inventive concept can be combined in whole or in part to obtain other embodiments that still fall within the scope of the inventive concept as defined in the appended claims.

[0024] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0025] Although some aspects have been described in the context of an apparatus, a system, or a device, it is clear that these aspects also represent a description of a method of operating such an apparatus, system, or device.

[0026] Other embodiments and advantages of the inventive concept will be described below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 An embodiment of a system according to the inventive concept is schematically illustrated;

[0028] Figure 2 Another embodiment of a system according to the inventive concept is schematically illustrated; and

[0029] Figure 3 A microscope having a microscope stage according to the inventive concept is schematically illustrated, the microscope stage having a system. DETAILED DESCRIPTION

[0030] In the following, the accompanying drawings are fully described, and the same reference numerals denote the same or at least structurally identical components.

[0031] Figure 1 A part of the microscope stage 160 is schematically illustrated, more precisely, a part of the cryo-stage 362 as shown in more detail in Figure 3 . The cryo-stage is adapted to receive a sample, typically a vitrified sample on a sample grid, examine the sample by optical microscopy to locate relevant areas of the sample, and then observe these areas in more detail in a cryo-electron microscope.

[0032] Figure 1 The cryo-stage of includes a system 100 according to the inventive concept. The system 100 includes a reservoir 110 filled with a cryogenic liquid (in particular liquid nitrogen) up to a filling level X. In principle, the reservoir 110 is configured to be filled with the cryogenic liquid at least up to a maximum filling level X max . A probe 120 is provided in the reservoir 110, the probe 120 including a probe body 122 and a temperature-related sensor 128. The probe body 122 extends from a (lower) first position 126 (at least) to a (higher) second position 124, the second position 124 being above the maximum filling level X max . The system 100 further includes a controller 250 (see Figure 2 ), which is used to control the filling level X of the reservoir 110 according to the sensor signal of the temperature-related sensor 128 such that the filling level X remains constant.

[0033] As can be seen from Figure 1It can be seen that, in terms of the height dimension of the reservoir 110, the second position 124 is laterally offset relative to the first position 126 (and vice versa). In other words, the probe body 122 diagonally extends through at least a portion of the reservoir in terms of the width dimension of the reservoir. In Figure 1 the projection on the vertical plane of the drawing plane, the probe body 122 is shown over the entire length of the probe body 122. In the projection on the vertical plane perpendicular to the drawing plane, the probe body 122 will be visible over a shorter length (in Figure 1 the embodiment, the projected length will be approximately equal to the height of the reservoir). In the projection on the horizontal plane, the length of the probe body 122 corresponds to the lateral displacement of the second position 124 relative to the first position 126. In the above description, the term "length of the probe body" refers to the length of the portion of the probe body from the first position 126 to the second position 124.

[0034] The probe body 122 diagonally passes through most of the reservoir 110 in terms of the width dimension of the reservoir 110, increasing the sensing surface of the thermally conductive probe body 122, thereby increasing the dynamic range and accuracy of the temperature-related sensor signal related to the filling level X.

[0035] Minimum filling level X min can be set arbitrarily in principle and can correspond to the height of the first position 126. In Figure 1 the illustrated embodiment, the first position 126 on the probe body 122 is lower than the minimum filling level X min .

[0036] Figure 1 The system 100 is adapted to keep the filling level X constant by measuring the temperature (or any other temperature-related signal) at the second position 124. Since this temperature decreases as the filling level of the cryogenic liquid in the reservoir 110 increases, calibration of the sensor signal can be used to determine the filling level X. Once the desired filling level is reached, the temperature-related sensor is calibrated at the corresponding temperature at the second position 124, and this temperature is used as the setpoint for the controller 250 (see Figure 2 ). Once the actual value of the measured temperature (or another temperature-related sensor signal) exceeds the setpoint, the filling level X of the reservoir increases until the setpoint is reached again.

[0037] Figure 2Schematically shows the arrangement of the microscope stage 160 (in particular the cryo-stage 362), the controller 250, and the flow regulator 240 that implements the above-described adjustment method. The temperature-related sensor 128 is connected to the controller 250 to transmit a temperature-related sensor signal to the controller 250. The controller determines whether the actual value of the sensor signal exceeds a set value, for example, whether it exceeds a predetermined threshold of the set value. If so, the controller 250 activates the flow regulator 240 to pump liquid nitrogen from the liquid nitrogen refill container into the supply line 230, as Figure 2 shown. This will cause the temperature-related sensor signal to drop below the threshold again, enabling the controller 250 to cause the flow regulator 240 to prevent further pumping of liquid nitrogen.

[0038] In this embodiment, the probe body 122 is made of steel, in particular X5CrNi18-10. As can be seen from Figure 1 it, the probe body 122 is strengthened over a length above the maximum filling level X max This further increases the sensing area of the probe 120. The temperature-related sensor 128 in this embodiment is a PT1000-sensor, whose resistance depends on the temperature at the second position 124. Figure 1 The arrangement shown results in an approximately linear correlation between the sensor signal and the filling level X.

[0039] Figure 3Schematically shows a microscope 300 including a microscope stage 160, and the microscope stage 160 includes the above-mentioned system 100. The microscope 300 includes a microscope stage 160, and the microscope stage 160 includes a cryo-stage 362 and a positioning stage 364 for moving the cryo-stage 362 in the x-y-z directions. The microscope 300 further includes a microscope objective 340 and an eyepiece 330. The microscope 300 further includes other components that are not described herein because they are not very relevant to the inventive concept. The microscope 300 is a cryo-optical microscope, and the cryo-optical microscope has a cryo-stage 362, and the cryo-stage 362 is used to receive a sample to be examined under the microscope 300. For this purpose, a shuttle 400 can be connected to the cryo-stage 362. The shuttle 400 contains a vitrified sample on a transfer rod. In its retracted position, the vitrified sample is at a loading position at low temperature. The shuttle 400 can be connected to the cryo-stage 362, and by sliding the transfer rod along the longitudinal direction of the transfer rod, the sample can be transferred into the cryo-stage 362 under cryogenic conditions. In the cryo-stage 362, the sample (usually a sample on a sample holder) is at its inspection position. To maintain the necessary cryogenic conditions, the system 100 is operated as described above. The Dewar vessel 500 stores a sufficient amount of liquid nitrogen, and the liquid nitrogen can be pumped into the supply line 230 through a flow regulator 240. The supply line 230 is insulated to avoid heating the liquid nitrogen inside the supply line 230. The flow regulator 240 is operated according to the principle explained in combination with Figure 2 to pump a necessary amount of liquid nitrogen from inside the Dewar vessel 500 into a reservoir 110 inside the cryo-stage 362 to keep the filling level inside the reservoir 110 constant. By examining the sample using the microscope 300, an area of interest in the nanometer range can be identified within a small volume (millimeter range) of the sample. Then, the area of interest is traced back in a cryo-electron microscope for high-resolution imaging.

[0040] Reference numeral

[0041] 100 System

[0042] 110 Reservoir

[0043] 112 Bottom side

[0044] 120 Probe

[0045] 122 Probe body

[0046] 124 Second position

[0047] 126 First position

[0048] 128 Temperature-related sensor

[0049] 160 Microscope stage

[0050] 230 Supply pipeline

[0051] 240 Flow regulator

[0052] 250 Controller

[0053] 300 Microscope

[0054] 330 Eyepiece

[0055] 340 Microscope objective

[0056] 362 Cryogenic stage

[0057] 364 Positioning stage

[0058] 400 Shuttle

[0059] 500 Dewar vessel

[0060] α Tilt angle

[0061] X Filling level

[0062] X min Minimum filling level

[0063] X max Maximum filling level.

Claims

1. A system (100) for controlling the filling level (X) of a liquid-filled reservoir (110), the system (100) comprising: The reservoir (110), the reservoir (110) being configured to be filled with a liquid up to at least a maximum filling level (X max ); Probe (120), the probe (120) includes a probe body (122), at least a portion of the probe body (122) extends from a first position (126) to a second position (124) in the height dimension of the liquid reservoir (110), the first position (126) is lower than the second position (124), and the second position (124) is higher than the maximum filling level (X max ), and the probe (120) further includes a temperature-related sensor (128), the temperature-related sensor (128) is configured to generate a sensor signal based on the temperature at the second position (124), the temperature depending on the filling level (X); and A controller (250) for controlling the filling level (X) of the reservoir (110) according to the sensor signal such that the filling level (X) remains constant.

2. The system (100) according to claim 1, wherein, The first position (126) on the probe body (122) is located at or below the minimum filling level (X min ) min .

3. The system (100) according to claim 1 or 2, wherein, The first position (126) is laterally offset relative to the second position (124).

4. The system (100) according to claim 3, wherein, The probe body (122) extends through most of the reservoir (110) in the width dimension of the reservoir (110).

5. The system (100) according to claim 3 or 4, wherein, The angle of inclination (α) of the probe body (122) relative to the bottom side (112) of the reservoir (110) is equal to or less than 65°, 55°, 45°, 35° or 25°.

6. The system (100) according to any one of the preceding claims, wherein, The probe body (122) is in the form of a beam, rod, wire or sheet.

7. The system (100) according to any one of the preceding claims, wherein, The probe body (122) is made of metal or steel.

8. The system (100) according to any one of the preceding claims, wherein, The probe body (122) is reinforced in the portion above the maximum filling level (X max ).

9. The system (100) according to any one of the preceding claims, wherein, The temperature-related sensor (128) is mounted on the probe body (122) at the second position (124) of the probe body (122).

10. The system (100) according to claim 9, wherein, The temperature-related sensor (128) is a resistance thermometer.

11. The system (100) according to any one of the preceding claims, the system (100) further comprising: A supply line (230) for supplying liquid into the reservoir (110); A flow regulator (240) for regulating the flow rate of the liquid entering the reservoir (110); and The controller (250) is connected to the temperature-related sensor (128) and the flow regulator (240) for controlling the filling level (X) of the reservoir (110) by adjusting the flow rate of the liquid entering the reservoir (110) according to the sensor signal.

12. The system (100) according to any one of the preceding claims, wherein, The liquid is a cryogenic liquid.

13. A microscope stage (160) of a microscope (300), the microscope stage (160) comprising the system (100) according to any one of the preceding claims.

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