A liquid nitrogen level control device and method for ultralow temperature milling and imaging

Abstract

The application relates to a liquid nitrogen liquid level control device and method for ultralow-temperature milling and imaging, which comprises a position-fixed milling module and imaging module, a liquid nitrogen storage tank, a buffer tank and a sample groove, the buffer tank is communicated with a liquid outlet pipe and a liquid suction pipe, the liquid outlet pipe and the liquid suction pipe are respectively used for filling liquid nitrogen into the sample groove and sucking liquid nitrogen, the sample groove is movably arranged, a temperature sensor is fixed on the milling module, one end of the liquid suction pipe located outside the buffer tank is a liquid suction end, the height of the liquid suction end is adjustable and is lower than the height of the temperature sensor. The device judges whether the liquid level reaches the milling module during the liquid nitrogen filling process through the fixed temperature sensor, so that the requirement that the sample surface is lower than the liquid nitrogen liquid level during milling is met. During the liquid nitrogen sucking process, the height of the liquid suction end can be directly used to control the liquid nitrogen liquid level during the liquid nitrogen sucking process, the liquid nitrogen use rate can be improved, and the requirement that the liquid nitrogen liquid level is lower than the sample surface during imaging is met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a liquid nitrogen liquid level control device and method for ultra-low temperature milling and imaging. BACKGROUND

[0002] At normal pressure, the temperature of liquid nitrogen is -196℃, and liquid nitrogen belongs to refrigerant. Due to its low temperature, strong stability, non-toxicity and non-pollution, liquid nitrogen is widely used in many fields such as industry, medicine, biology and the like, for example, rapid freezing of food, storage and freezing of biological tissues, medical experiment removal of pathological tissues, cryotherapy and the like.

[0003] In the research on the influence of different parameters of ultra-low temperature milling on the surface quality of the processed material, the commonly used experimental methods are as follows: a processed workpiece pre-cooled by liquid nitrogen or immersed in liquid nitrogen in real time is clamped on a customized container type clamp for milling, and the milling environment is detected and adjusted on line through a temperature sensor at the milling position, and the judgment of real-time liquid supplement is made, wherein the liquid nitrogen supplement in real time is manually poured, which cannot be effectively automated; or a liquid nitrogen internal cooling tool is used in combination with an external cooling nozzle to maintain a low temperature environment during milling, and a temperature sensor is used to measure the low temperature at the milling position, and after the milling is completed, the workpiece is unloaded and placed under a microscope or an electron microscope for imaging, and the surface texture is observed, wherein the method for maintaining a low temperature is to use a liquid nitrogen pipeline to locally spray low-temperature fluid, and the pipeline is difficult to avoid problems such as long path, large heat leakage, large pipeline liquid outlet pressure, uncontrollable flow rate and spatter.

[0004] In the biomedical field, the requirements for ultra-low temperature milling and imaging of tissue blocks are different from those for ordinary mechanical materials, especially when the tissue block is imaged layer by layer, in order to avoid the influence of milling heat on the surface quality of the sample, the tissue block needs to be immersed in liquid nitrogen under the milling state, and in deep low temperature imaging, the objective lens used for imaging is generally an air lens with a long working distance to avoid damage to the objective lens caused by low temperature, so the liquid level of liquid nitrogen needs to be lower than the sample surface during imaging, and in order to avoid the influence of the milling debris on the sample surface or the ice crystals formed on the sample surface due to the rapid cooling of water vapor in the air, flushing needs to be performed before imaging; and the thickness of each layer of sample is usually microns, and if the liquid level is too low during imaging, the temperature of the sample surface will be too high, which will affect the fluorescence intensity in the sample, resulting in weak fluorescence intensity during imaging and affecting the imaging result. Therefore, in order to complete the layer-by-layer section imaging, the liquid level of liquid nitrogen needs to be controlled with high precision at a position higher than the sample surface and a position lower than the sample surface, and the current control scheme for liquid nitrogen supplement under ultra-low temperature milling cannot meet the scene requirements of the layer-by-layer section fluorescence imaging of the tissue block. SUMMARY

[0005] The technical problems to be solved by the present application are to provide a liquid nitrogen level control device and method for ultra-low temperature milling and imaging, to realize buffer tank liquid supplement, sample groove liquid supplement control level and liquid nitrogen recovery, to improve the efficiency of ultra-low temperature milling and imaging and the stability of liquid nitrogen liquid, and to solve the problem of accurate control of liquid nitrogen level in the biomedical field during ultra-low temperature milling and imaging.

[0006] The specific solutions provided by the present application are as follows:

[0007] The present application provides a liquid nitrogen level control device for ultra-low temperature milling and imaging, comprising a fixed milling module and an imaging module, and further comprising a liquid nitrogen storage tank, a buffer tank and a sample groove, wherein the liquid nitrogen storage tank is in communication with the buffer tank for providing liquid nitrogen to the buffer tank, the buffer tank is in communication with a liquid outlet pipe and a liquid suction pipe for respectively filling and sucking liquid nitrogen into the sample groove, and the sample groove is movably arranged, a temperature sensor is fixed on the milling module, and the liquid suction end of the liquid suction pipe located outside the buffer tank is adjustable in height and lower than the height of the temperature sensor.

[0008] The device can determine whether the liquid level reaches the milling module during liquid nitrogen filling through the temperature sensor fixed on the milling module, thereby meeting the requirement that the sample surface is lower than the liquid level of liquid nitrogen during milling, improving the efficiency of ultra-low temperature milling and imaging, and solving the technical problem of low efficiency of manual pouring. When liquid nitrogen is sucked, the height of the liquid suction end is adjustable, and in combination with the height of the sample groove, the height of the liquid suction end can be directly used to control the liquid level of liquid nitrogen during liquid nitrogen suction, and when the liquid level of liquid nitrogen is lower than the height of the liquid suction end, the liquid nitrogen suction is stopped. The liquid suction pipe can not only be used for recovering liquid nitrogen to improve the utilization rate of liquid nitrogen, but also meet the requirement that the liquid level of liquid nitrogen is lower than the sample surface during imaging.

[0009] Further, a liquid suction port is formed on the buffer tank, the liquid suction pipe comprises a main pipe and an end pipe, the main pipe is located in the buffer tank and fixed with the liquid suction port, the end pipe is located outside the buffer tank and detachably connected with the liquid suction port, and the liquid suction end is one end of the end pipe away from the liquid suction port.

[0010] Further, a filter is arranged at one end of the liquid suction pipe located outside the buffer tank.

[0011] Further, the height of the temperature sensor is h1, the height of the liquid suction end is h2, and 4mm≤h2-h1≤6mm.

[0012] Further, a three-dimensional translation stage is further included, and the sample groove is fixed on the three-dimensional translation stage.

[0013] Further, the buffer tank top is provided with an air extraction device, which further comprises a hose, a heat exchanger and an air extraction pump, and the hose is connected with the buffer tank, the heat exchanger and the air extraction pump in sequence.

[0014] Further, the buffer tank top is provided with an air extraction device, which further comprises a hose, a heat exchanger and an air extraction pump, and the hose is connected with the buffer tank, the heat exchanger and the air extraction pump in sequence.

[0015] Based on the technical scheme of the present application, the following beneficial effects are achieved:

[0016] (1) Based on the liquid nitrogen level control device for ultra-low temperature milling and imaging, the buffer tank liquid supplementing, sample groove liquid supplementing and liquid extraction control level can be automatically realized in time, the liquid nitrogen level in the sample groove can be quickly and stably adjusted during the milling and imaging of the sample, the requirements that the sample surface is lower than the liquid nitrogen level during the milling and the sample surface is higher than the liquid nitrogen level during the imaging are met, the efficiency of the ultra-low temperature milling and imaging is improved, and the technical problem of low efficiency of manual pouring is solved; the buffer tank body is used as a liquid supplementing and recycling device, the liquid supplementing and extraction of the liquid nitrogen in the external container can be realized, the liquid nitrogen recycling is realized, and the utilization rate of the liquid nitrogen is improved.

[0017] (2) When the buffer tank supplements the liquid to the sample groove, the entire buffer tank interior can maintain normal pressure through the air outlet, the liquid is discharged by gravity, and when the liquid nitrogen storage tank is used to supplement the liquid to the sample groove, the liquid supplementing can also be carried out at normal pressure, so that the liquid nitrogen discharging is stable and less boiling, and the splashing during the liquid supplementing is reduced or avoided; in the prior art, the liquid is usually pumped to the sample groove by using a dewar bottle pressure, an external pressure source or internal heating is needed to increase the pressure, the splashed fluid has a large gas content, and the liquid cannot be stably discharged, and the use of the buffer tank body can avoid the direct liquid supplementing through a long pipeline, reduce the gas content in the splashed fluid, and further reduce the liquid nitrogen splashing during the liquid supplementing of the sample groove.

[0018] (3) The liquid supplementing is carried out on the sample groove through the liquid discharging pipe, the liquid extraction is carried out through the liquid extraction pipe, the liquid supplementing amount is controlled through the temperature sensor, and the liquid extraction amount is controlled through the height of the translation table relative to the liquid extraction end, so that the liquid nitrogen level in the sample groove is strictly controlled to meet the specific experimental requirements.

[0019] The present application also provides a liquid nitrogen level control method for ultra-low temperature milling and imaging, which comprises the following steps:

[0020] S1, liquid supplementing: the sample groove is moved to the liquid discharging pipe and the sample surface in the sample groove is lower than the height of the temperature sensor, the liquid nitrogen in the buffer tank enters the sample groove through the liquid discharging pipe, and when the temperature sensor detects that the temperature is not higher than a third set value, the liquid discharging pipe stops the liquid supplementing, and the third set value ranges from -190 DEG C to -196 DEG C;

[0021] S2, milling: the sample slot moves to the milling module, the milling module mills the sample surface in the sample slot to expose a new layer of sample surface;

[0022] S3, pumping: adjust the height of the pumping end, the sample slot moves to the pumping tube, the liquid nitrogen in the sample slot enters the buffer tank through the pumping tube, when the liquid nitrogen level in the sample slot is lower than the height of the pumping end, pumping stops;

[0023] S4, flushing: the sample slot moves to the liquid outlet tube, the liquid nitrogen in the buffer tank enters the sample slot through the liquid outlet tube, and the new layer of sample surface in the sample slot is flushed;

[0024] S5, imaging: the sample slot moves to the imaging module, the imaging module images the new layer of sample surface;

[0025] S6, repeat S1-S5 until the required number of layers of imaging is completed.

[0026] The method can be realized based on the aforementioned liquid nitrogen level control device for ultra-low temperature milling and imaging, and is used for cyclically and accurately controlling the liquid nitrogen level when performing multi-layer milling and imaging in the biomedical field. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Structure diagram of the device for ultra-low temperature milling and imaging based on the application Figure 1 .

[0028] Figure 2 Fluorescence imaging diagram of the sample under different liquid levels.

[0029] Figure 3 SEM diagram of the sample surface before and after flushing.

[0030] Figure 4 Structure diagram of the device for ultra-low temperature milling and imaging based on the application Figure 2 .

[0031] Figure 5 Flowchart of the buffer tank liquid supplementing based on the application.

[0032] Figure 6 Flowchart of the liquid addition / flushing to the sample slot through the liquid outlet tube based on the application.

[0033] In the attached diagram: 1. Liquid nitrogen storage tank; 11. Liquid replenishment solenoid valve; 2. Buffer tank; 21. Liquid outlet pipe; 211. Pneumatic valve; 212. Nozzle; 22. Liquid extraction pipe; 221. Liquid extraction port; 222. Main pipe; 223. End pipe; 224. Filter; 23. Hose; 231. Air extraction solenoid valve; 24. Heat exchanger; 25. Air extraction pump; 26. Exhaust port; 261. Exhaust solenoid valve; 27. Liquid level gauge; 3. Sample tank; 4. Temperature sensor; 5. Three-dimensional translation stage; 6. Milling module; 7. Imaging module. Detailed Implementation

[0034] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0035] The present invention will now be described with reference to the figures and specific embodiments.

[0036] like Figure 1 As shown, the liquid nitrogen level control device for cryogenic milling and imaging based on the present invention includes a milling module and an imaging module with fixed positions, and further includes: a liquid nitrogen storage tank, a buffer tank and a sample tank. The liquid nitrogen storage tank is connected to the buffer tank and is used to supply liquid nitrogen to the buffer tank. The buffer tank is connected to an outlet pipe and a suction pipe, which are used to add liquid nitrogen to the sample tank and to draw liquid nitrogen out, respectively. The sample tank is movable. A temperature sensor is fixed on the milling module. The end of the suction pipe located outside the buffer tank is the suction end. The height of the suction end is adjustable and lower than the height of the temperature sensor.

[0037] The liquid nitrogen level control device for cryogenic milling and imaging based on this invention can automatically and promptly control the liquid level by replenishing the buffer tank and the sample tank, allowing for rapid and stable adjustment of the liquid nitrogen level in the sample tank during milling and imaging. A temperature sensor fixed to the milling module determines whether the liquid nitrogen level has reached the milling module during addition, thus meeting the requirement that the sample surface be below the liquid nitrogen level during milling, improving the efficiency of cryogenic milling and imaging, and solving the technical problem of low efficiency with manual pouring. When aspirating liquid nitrogen, an adjustable suction end is provided. Combined with the height of the sample tank, the height of the suction end can be directly used to control the liquid nitrogen level during suction. Suction is stopped when the liquid nitrogen level is below the height of the suction end. The suction tube of this invention not only recovers liquid nitrogen and improves its utilization rate but also meets the requirement that the liquid nitrogen level be below the sample surface during imaging.

[0038] In this invention, the height settings of the temperature sensor and the suction end enable higher precision liquid level control. For example, by setting the temperature sensor 2mm above the milling point, liquid addition can be stopped when the temperature detected by the sensor is around -196℃. At this point, the liquid nitrogen level in the sample tank is 2mm above the milling point, and since the thickness of each milling operation is on the micrometer scale and negligible, the liquid nitrogen level will remain approximately 2mm above the sample surface during milling. Simultaneously, by setting the suction end 5mm below the temperature sensor, the liquid nitrogen level will be approximately 3mm below the sample surface after suction. This satisfies the requirement that the liquid nitrogen level be below the sample surface during imaging while maintaining the sample surface temperature, thereby ensuring the fluorescence intensity during imaging. Figure 2 The images show weak fluorescence intensity imaging (a) when the liquid nitrogen level is significantly lower than the sample surface and strong fluorescence intensity imaging (b) when the liquid nitrogen level is only slightly lower than the sample surface. In contrast, existing technologies using liquid level sensors or other methods require larger changes in liquid level to detect these changes. Therefore, this invention offers higher precision in controlling the liquid nitrogen level. Furthermore, this invention uses a buffer tank as a replenishment and recovery device, which is smaller, easier to transfer, and suitable for the biomedical imaging field. In existing technologies, liquid nitrogen is typically added directly from the storage tank to the milling area, for example, using a Dewar flask pressure pump. This requires an external pressure source or internal heating and pressurization, resulting in a high gas content in the splashed fluid and unstable liquid output. Using a buffer tank avoids direct addition via long pipes, reducing the gas content in the splashed fluid and further minimizing liquid nitrogen splashing during the sample tank replenishment process in this invention.

[0039] In the cryogenic milling and imaging scenario of this invention, the system employs a tomographic imaging method, where milling and imaging alternate, and the sample height continuously decreases. Therefore, the liquid nitrogen level required for each layer of milling and imaging is different relative to the bottom of the sample tank. After each layer of milling and imaging, the sample can be raised by one layer to ensure that the liquid nitrogen level required for the next layer of milling and imaging is fixed relative to the milling module. Therefore, a temperature sensor is fixed at a certain position above the milling module to detect whether the liquid level is above the sample surface before milling begins. If, before milling, the sample tank moves to the milling position, the temperature reading from the temperature sensor is higher than the set temperature value, it indicates that the liquid nitrogen level in the sample tank is lower than the sample surface. In this case, liquid nitrogen needs to be added to the sample tank through the outlet pipe to cool it down until the temperature sensor value is no higher than the set temperature value, then the addition of liquid nitrogen is stopped, and the milling operation is then performed. The liquid nitrogen temperature is -196℃, preferably -190 to 195℃. The temperature sensor can be a T-type thermocouple temperature sensing wire sensor with a response time of less than 0.1s.

[0040] Similarly, for the reasons mentioned above, the suction end of the suction tube is fixed at a certain position below the milling cutter module to ensure that the liquid nitrogen level is below the sample surface before imaging begins. During milling, to avoid the milling heat affecting the sample surface quality, the liquid level needs to be higher than the sample surface. During imaging, the objective lens used is generally a long working distance air lens to avoid damage to the objective lens due to low temperature; therefore, the liquid level needs to be lower than the sample surface. When the liquid level drops, to prevent debris and ice crystals from settling on the sample surface and affecting the imaging effect, the sample surface needs to be rinsed, such as... Figure 3 The image shows the samples before and after milling (a) and after rinsing (b) under the same conditions. As can be seen, a large amount of debris settles on the sample surface after the liquid level drops, thus requiring a rinsing process. The specific rinsing process is as follows: After the milling operation, liquid is added to the sample tank again through the outlet pipe to rinse the sample surface. During rinsing, the sample tank can be moved to ensure the rinsing area covers the entire sample surface. Therefore, placing the outlet and the extraction pipe adjacent to each other allows them to be as close to the sample tank as possible simultaneously, reducing the length of the outlet pipe, the extraction pipe, and the movement range of the sample tank during rinsing, thereby reducing heat leakage and simplifying the rinsing process.

[0041] like Figure 4 As shown, in some embodiments, the buffer tank has a liquid extraction port. The extraction tube includes a main pipe and an end pipe. The main pipe is located inside the buffer tank and fixed to the extraction port, while the end pipe is located outside the buffer tank and detachably connected to the extraction port. The extraction end is the end of the outer pipe furthest from the extraction port. Specifically, the end pipe and the extraction port can be detachably connected via a threaded connection. Furthermore, PTFE tape can be used at the connection point to enhance sealing. The detachable design of the end pipe allows for the selection of end pipes of different lengths to be connected to the outside of the buffer tank. Therefore, the height of the extraction end can be adjusted, meeting the liquid level control requirements of different application scenarios without requiring the sample tank to be moved up and down.

[0042] Furthermore, a filter is installed at one end of the suction tube outside the buffer tank. Since some debris will remain in the liquid nitrogen after milling in the sample cell, the liquid nitrogen in the sample cell can be filtered through the filter at the suction end when using the suction tube to draw liquid nitrogen, so that the liquid nitrogen returned to the buffer tank is purer and can be used for the next liquid nitrogen filling.

[0043] Further, the height of the temperature sensor is h1, the height of the liquid suction end is h2, and 4mm≤h2-h1≤6mm. The height of the temperature sensor is used to control the liquid level during liquid nitrogen filling, and the height of the liquid suction end cooperates with the position of the sample groove to control the liquid level of the liquid nitrogen during suction. When the height of the sample groove in the vertical direction is constant, the height difference between the liquid suction end and the temperature sensor within the above range can accurately control the liquid level difference between the liquid nitrogen filling and suction within the range of 4mm-6mm, which can meet the temperature control requirements of the sample block in the biomedical field during one layer of milling and imaging. Of course, in other embodiments, the height of the sample groove in the vertical direction can also be controlled to change: for example, when milling, the height of the sample surface in the sample groove is h3, then the temperature sensor can be installed 1mm-2mm higher than the sample surface, then when filling the liquid nitrogen, the liquid level of the liquid nitrogen can be controlled to be 1mm-2mm higher than the sample surface to stop. At the same time, the height of the liquid suction end is set to be 3mm-5mm lower than the sample surface, so that when the liquid nitrogen is sucked, the liquid level of the liquid nitrogen can be controlled to be 3mm-5mm lower than the sample surface to stop.

[0044] By setting the height of the temperature sensor and the liquid suction end in the present application, the control of the liquid level of the liquid nitrogen can achieve a precision of millimeter level, which can meet the requirements of micron-level milling and imaging operation of the sample in the biomedical field.

[0045] Further, the liquid nitrogen liquid level control device for ultra-low temperature milling and imaging also includes a three-dimensional translation stage, and the sample groove is fixed on the three-dimensional translation stage. Therefore, the sample groove can be translated between the liquid outlet pipe, the milling module, the liquid suction pipe and the imaging module; at the same time, the sample groove can also move in the vertical direction, i.e. after each pair of sample surfaces is milled and imaged, the sample can be lifted for milling and imaging of new sample surfaces, and during liquid suction, the sample groove can also be moved in the vertical direction to cooperate with the position of the liquid suction end to accurately control the liquid level of the liquid nitrogen during suction.

[0046] Specifically, the milling module adopts an existing structure, such as a high-speed precision spindle head matched with a direct coupling servo motor, and the end of the spindle head clamps a face milling cutter through a precision level chuck, and the motor drives the milling cutter on the spindle to rotate, which cooperates with the three-dimensional translation stage to mill the sample surface. Specifically, the imaging module also adopts an existing structure, such as a commercial body microscope with macro zoom function and a camera, which can perform wide-field imaging or line scanning imaging with different resolutions according to different sizes of samples and actual application requirements. Specifically, the three-dimensional translation stage can be fixed on a marble table, the milling module and the imaging module can be fixed on the marble table gantry, and the sample groove is fixed above the three-dimensional translation stage.

[0047] Further, the buffer tank top is provided with an exhaust port, and the sample groove is also provided with an open port and uses a vacuum sandwich heat preservation structure, and the exhaust port is provided with an exhaust electromagnetic valve, so that the exhaust electromagnetic valve is opened, and when the buffer tank body adds liquid to the sample groove, the entire buffer tank body can maintain normal pressure through the exhaust port, and the sample groove is also at normal pressure, and the liquid is discharged by gravity, and the sample groove is filled with liquid at normal pressure, so that the liquid nitrogen is discharged smoothly and less boiling, and the splashing during filling is reduced or avoided. At the same time, a pneumatic valve is arranged on the liquid outlet pipe, and a nozzle is detachably arranged at one end of the liquid outlet pipe outside the buffer tank, for example, the nozzle is threadedly connected with the liquid outlet. The low-temperature electromagnetic valve commonly used in industry needs to be used under pressure, and the heat insulation effect is not good, and the gasification amount is large, and the pneumatic valve is arranged on the liquid outlet pipe, so that the liquid discharge can be controlled under normal pressure, the liquid discharge is smooth, and the splashing is reduced; and the pneumatic valve has a rapid response time, that is, it can be opened and closed immediately, the heat insulation effect is good, and the liquid nitrogen gasification amount is reduced. The commonly used filling method has long pipeline, and pressure regulating valve flow meter and other parts are installed, so that the heat leakage amount is large, the liquid nitrogen gasification is serious, and the liquid is mostly sprayed, which is not suitable for laboratory small flow liquid supplementing. The device based on the application discharges liquid by gravity under normal pressure, so the liquid nitrogen gasification amount is small, the boiling is not serious, the liquid discharge is relatively smooth, and the liquid flow can be adjusted by arranging nozzles with different diameters, which is suitable for laboratory open small volume container liquid supplementing.

[0048] Further, the buffer tank top is provided with an exhaust port, and the sample groove is also provided with an open port and uses a vacuum sandwich heat preservation structure, and the exhaust port is provided with an exhaust electromagnetic valve, so that the exhaust electromagnetic valve is opened, and when the buffer tank body adds liquid to the sample groove, the entire buffer tank body can maintain normal pressure through the exhaust port, and the sample groove is also at normal pressure, and the liquid is discharged by gravity, and the sample groove is filled with liquid at normal pressure, so that the liquid nitrogen is discharged smoothly and less boiling, and the splashing during filling is reduced or avoided. At the same time, a pneumatic valve is arranged on the liquid outlet pipe, and a nozzle is detachably arranged at one end of the liquid outlet pipe outside the buffer tank, for example, the nozzle is threadedly connected with the liquid outlet. The low-temperature electromagnetic valve commonly used in industry needs to be used under pressure, and the heat insulation effect is not good, and the gasification amount is large, and the pneumatic valve is arranged on the liquid outlet pipe, so that the liquid discharge can be controlled under normal pressure, the liquid discharge is smooth, and the splashing is reduced; and the pneumatic valve has a rapid response time, that is, it can be opened and closed immediately, the heat insulation effect is good, and the liquid nitrogen gasification amount is reduced. The commonly used filling method has long pipeline, and pressure regulating valve flow meter and other parts are installed, so that the heat leakage amount is large, the liquid nitrogen gasification is serious, and the liquid is mostly sprayed, which is not suitable for laboratory small flow liquid supplementing. The device based on the application discharges liquid by gravity under normal pressure, so the liquid nitrogen gasification amount is small, the boiling is not serious, the liquid discharge is relatively smooth, and the liquid flow can be adjusted by arranging nozzles with different diameters, which is suitable for laboratory open small volume container liquid supplementing.

[0049] A pneumatic valve is arranged on the liquid outlet pipe, and a nozzle is detachably arranged at one end of the liquid outlet pipe outside the buffer tank, for example, the nozzle is threadedly connected with the liquid outlet. The low-temperature electromagnetic valve commonly used in industry needs to be used under pressure, and the heat insulation effect is not good, and the gasification amount is large, and the pneumatic valve is arranged on the liquid outlet pipe, so that the liquid discharge can be controlled under normal pressure, the liquid discharge is smooth, and the splashing is reduced; and the pneumatic valve has a rapid response time, that is, it can be opened and closed immediately, the heat insulation effect is good, and the liquid nitrogen gasification amount is reduced. The commonly used filling method has long pipeline, and pressure regulating valve flow meter and other parts are installed, so that the heat leakage amount is large, the liquid nitrogen gasification is serious, and the liquid is mostly sprayed, which is not suitable for laboratory small flow liquid supplementing. The device based on the application discharges liquid by gravity under normal pressure, so the liquid nitrogen gasification amount is small, the boiling is not serious, the liquid discharge is relatively smooth, and the liquid flow can be adjusted by arranging nozzles with different diameters, which is suitable for laboratory open small volume container liquid supplementing.

[0050] Further, the buffer tank adopts a vacuum sandwich heat preservation structure, and a cover plate at the upper end of the buffer tank is sealingly connected with the tank body through a flange and a gasket. The buffer tank and the sample tank both adopt a vacuum sandwich heat insulation, which can reduce the gasification amount and loss of liquid nitrogen and improve the stability of the liquid level of the liquid nitrogen.

[0051] The liquid nitrogen level control device for ultra-low temperature milling and imaging based on the embodiment of the present application further comprises a control device (not shown in the figure), and the control device comprises a first control unit and a second control unit.

[0052] As shown in Figure 4 , the first control unit is used for closed-loop control of automatic supplement of liquid nitrogen in the buffer tank, and the first control unit is electrically connected with a liquid supplement electromagnetic valve, a gas extraction electromagnetic valve, a gas discharge electromagnetic valve and a liquid level meter. Firstly, the value of the liquid level meter is read as the liquid level of the buffer tank. When the liquid level is lower than a first set value, the gas extraction electromagnetic valve is controlled to be closed, the gas discharge electromagnetic valve is controlled to be opened, and the liquid supplement electromagnetic valve is controlled to be opened. The liquid nitrogen storage tank supplements liquid nitrogen to the buffer tank until the liquid level is not lower than a second set value, and then the liquid supplement electromagnetic valve is controlled to be closed.

[0053] As shown in Figure 5 , the second control unit is electrically connected with a three-dimensional translation stage, a pneumatic valve of a liquid outlet pipe and a temperature sensor. The second control unit is used for filling liquid nitrogen from the buffer tank into the sample tank, which can improve the liquid level of the liquid nitrogen in the sample tank, meet the milling demand, or flush the sample surface after milling. Firstly, the three-dimensional translation stage is controlled to move to drive the sample tank to move below the liquid outlet pipe. The value of the temperature sensor is read as the temperature of a specified height in the sample tank. When the temperature is higher than a third set value, the pneumatic valve of the liquid outlet pipe is controlled to be opened. The liquid outlet pipe fills liquid nitrogen into the sample tank until the temperature is not higher than the third set value, and then the pneumatic valve of the liquid outlet pipe is controlled to be closed. The third set value ranges from -190℃ to -196℃.

[0054] The present application further provides a liquid nitrogen level control method for ultra-low temperature milling and imaging, comprising the following steps:

[0055] S1, liquid filling: the sample tank moves to the liquid outlet pipe and the sample surface in the sample tank is lower than the height of the temperature sensor. The liquid nitrogen in the buffer tank enters the sample tank through the liquid outlet pipe. When the temperature sensor detects that the temperature is not higher than the third set value, the liquid outlet pipe stops filling liquid. The third set value ranges from -190℃ to -196℃.

[0056] S2, milling: the sample tank moves to the milling module, and the milling module mills the sample surface in the sample tank to expose a new layer of sample surface.

[0057] S3, pumping: the height of the pumping end is adjusted, the sample tank moves to the pumping pipe, the liquid nitrogen in the sample tank enters the buffer tank through the pumping pipe, and when the liquid nitrogen level in the sample tank is lower than the height of the pumping end, the pumping stops.

[0058] S4, flushing: the sample tank moves to the liquid outlet pipe, the liquid nitrogen in the buffer tank enters the sample tank through the liquid outlet pipe, and the new sample surface in the sample tank is flushed.

[0059] S5, imaging: the sample tank moves to the imaging module, and the imaging module images the new sample surface.

[0060] S6, repeat S1-S5 until the required number of layers of imaging is completed.

[0061] In S1 and S4, the buffer tank is in a normal pressure environment, and the liquid nitrogen enters the sample tank through the liquid outlet pipe by gravity.

[0062] In S3, the buffer tank is in a negative pressure environment, and the liquid nitrogen enters the buffer tank through the pumping pipe due to the negative pressure suction.

[0063] The specific working process of the device based on the embodiment of the application for milling and imaging is as follows: first, fix the sample in the sample tank, then move the sample tank to below the liquid outlet pipe through the three-dimensional translation table, then open the pneumatic valve and exhaust electromagnetic valve of the liquid outlet pipe, and add liquid to the sample tank under normal pressure until the temperature sensor at the fixed position of the milling module is not higher than the third set value, and then close the pneumatic valve of the liquid outlet pipe and stop adding liquid.

[0064] After the liquid is added, the pneumatic valve of the liquid outlet pipe is closed; then the milling module starts to mill the sample surface, which can be done by fixing the milling module and moving the sample relative to the milling module, and after milling a layer;

[0065] Before imaging, the liquid nitrogen level of the sample tank needs to be adjusted to be lower than the sample surface, so the sample tank can be moved to below the pumping port by the three-dimensional translation table, the exhaust device and the exhaust electromagnetic valve are opened, and the exhaust electromagnetic valve is closed, the liquid nitrogen in the sample tank above the pumping end is pumped back to the buffer tank by the negative pressure of the exhaust device. Before imaging, the sample tank can be adjusted to be below the liquid outlet pipe again, the pneumatic valve and exhaust electromagnetic valve of the liquid outlet pipe are opened, the liquid nitrogen in the buffer tank flows out, and the sample surface is flushed. After flushing, the pneumatic valve of the liquid outlet pipe is closed.

[0066] Then the camera images the sample surface, the three-dimensional translation table moves the sample tank to below the imaging module to start imaging, and finally the above steps are repeated until the required number of layers of imaging is completed.

[0067] Through the above steps, the liquid nitrogen level near the sample surface can be accurately controlled during multi-layer milling and imaging of the sample. In addition, when the number of imaging layers is large, the liquid nitrogen in the buffer tank will be insufficient, so the step of further supplementing liquid nitrogen can be added: opening the liquid supplementing electromagnetic valve at the communication position between the liquid nitrogen storage tank and the buffer tank to supplement the liquid nitrogen in the liquid nitrogen storage tank to the buffer tank.

[0068] Although the embodiments of the present application have been described in detail above, those skilled in the art can understand that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A liquid nitrogen level control device for ultralow temperature milling and imaging, for use in the field of biomedical imaging, comprising a fixed-in-place milling module and an imaging module, characterized in that, Also comprising: The liquid nitrogen storage tank is communicated with the buffer tank for providing liquid nitrogen to the buffer tank, the buffer tank is communicated with a liquid outlet pipe and a liquid suction pipe for filling and sucking liquid nitrogen into the sample tank respectively, the sample tank is movably arranged, a temperature sensor is fixed on the milling module, one end of the liquid suction pipe located outside the buffer tank is a liquid suction end, the height of the liquid suction end is adjustable and lower than the height of the temperature sensor; an air exhaust device is arranged on the top of the buffer tank, the air exhaust device further comprises a hose, a heat exchanger and an air exhaust pump, the hose is connected with the buffer tank, the heat exchanger and the air exhaust pump in sequence; an exhaust port is further arranged on the top of the buffer tank, the exhaust port is provided with an exhaust electromagnetic valve, the hose is provided with an air suction electromagnetic valve, the liquid outlet pipe is provided with a pneumatic valve, and a nozzle is detachably arranged on one end of the liquid outlet pipe located outside the buffer tank; a main pipe is arranged in the liquid suction pipe located in the buffer tank, and the height of the main pipe is higher than the height of the liquid level in the buffer tank; during liquid suction, the air exhaust device is used to reduce the pressure in the buffer tank to a negative pressure environment, and the height of the liquid suction end is used to control the liquid level of the liquid nitrogen during liquid suction; during liquid filling, the buffer tank is controlled to be under normal pressure through the exhaust port, and the liquid nitrogen enters the sample tank from the liquid outlet pipe by gravity; whether the liquid level during liquid filling reaches the milling module is judged by the fixed temperature sensor, so as to meet the requirement that the sample surface is lower than the liquid level of the liquid nitrogen during milling; the height of the liquid suction end and the temperature sensor is set to realize micron-level milling and imaging of the sample.

2. The liquid nitrogen level control device for ultralow-temperature milling and imaging according to claim 1, characterized in that, A liquid suction port is arranged on the buffer tank, the liquid suction pipe comprises a main pipe and an end pipe, the main pipe is located in the buffer tank and fixed with the liquid suction port, and the end pipe is located outside the buffer tank and detachably connected with the liquid suction port, and the liquid suction end is one end of the end pipe away from the liquid suction port.

3. The liquid nitrogen level control apparatus for ultralow-temperature milling and imaging according to claim 2, characterized by, A filter is arranged on one end of the liquid suction pipe located outside the buffer tank.

4. The liquid nitrogen level control apparatus for ultralow-temperature milling and imaging according to claim 2, characterized by, The height of the temperature sensor is h1, the height of the liquid suction end is h2, and 4mm≤h2-h1≤6mm.

5. The liquid nitrogen level control apparatus for ultralow-temperature milling and imaging according to claim 4, characterized by, A three-dimensional translation stage is further arranged, and the sample tank is fixed on the three-dimensional translation stage.

6. A method for liquid nitrogen level control for ultralow temperature milling and imaging, using the liquid nitrogen level control device according to claim 1, characterized in that, The method comprises the following steps: S1, liquid filling: the sample tank moves to the liquid outlet pipe, and the sample surface in the sample tank is lower than the height of the temperature sensor, the liquid nitrogen in the buffer tank enters the sample tank through the liquid outlet pipe, and when the temperature sensor detects that the temperature is not higher than a third set value, the liquid filling of the liquid outlet pipe is stopped, and the third set value ranges from-190℃ to-196℃; S2, milling: the sample tank moves to the milling module, the milling module mills the sample surface in the sample tank to expose a new layer of sample surface; S3, liquid suction: the height of the liquid suction end is adjusted, the sample tank moves to the liquid suction pipe, and the liquid nitrogen in the sample tank enters the buffer tank through the liquid suction pipe, and the liquid suction is stopped when the liquid level of the liquid nitrogen in the sample tank is lower than the height of the liquid suction end; S4, flushing: the sample tank moves to the liquid outlet pipe, and the liquid nitrogen in the buffer tank enters the sample tank through the liquid outlet pipe to flush the new layer of sample surface in the sample tank; S5, imaging: the sample tank is moved to the imaging module, and the imaging module images the new layer of sample surface; S6, repeating S1-S5 until the imaging of the required layers is completed.

7. The liquid nitrogen level control method for ultralow-temperature milling and imaging according to claim 6, characterized in that, During the liquid adding in S1 and the flushing in S4, the buffer tank is in a normal pressure environment, and the liquid nitrogen enters the sample tank from the liquid outlet pipe by gravity.

8. The liquid nitrogen level control method for ultralow-temperature milling and imaging according to claim 6, characterized in that, During the liquid pumping in S3, the buffer tank is in a negative pressure environment, and the liquid nitrogen enters the buffer tank from the liquid inlet pipe due to the negative pressure suction.

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