Freezing chip, freezing system, sample testing system and method

Abstract

The present disclosure discloses a freezing chip, freezing system, sample testing system and method. The freezing chip contacts a low-temperature cooling source and is used to freeze samples. The freezing chip comprises a heating layer and a heat-conducting layer arranged in sequence. The heating layer comprises at least one temperature control unit arranged on the heat-conducting layer. The heat generated by the temperature control unit is transferred to the low-temperature cooling source along the thickness direction of the heat-conducting layer. This technology can select specific times for freezing and thawing during in-situ observation and characterization of samples, and obtain a temperature greater than 10°C by designing the interface thermal resistance. 5 The freezing and heating speeds of 100°C / s ensure that the sample is not damaged. This is a major improvement for operations such as freezing, thawing, and in-situ microscopic observation of biological samples, and has great significance and broad application prospects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number "CN202011597320.1" filed on December 28, 2020, the entire contents of which are incorporated into this application as a whole. Technical Field

[0003] The present disclosure relates to the field of biomedical technology, and in particular to a freezing chip, a freezing system, a sample testing system and a method. Background Art

[0004] The rapid freezing and heating technology of biological samples has many important applications in the biomedical field, such as cell cryopreservation and revival, protein cryofixation and characterization, etc.

[0005] Current biological freezing technologies primarily include plunge freezing, jet freezing, and high-pressure freezing. Plunge freezing is the most commonly used sample preparation method in the industry. In plunge freezing, a sample holder (microgrid) holding the biological sample is fixed to the front end of a sample holder. A mechanical control rapidly inserts the sample into a cryogenic liquid, such as liquid ethane or liquid nitrogen, to freeze the sample. Jetting freezing involves transferring the sample holder to a specific position in a freezing chamber via a sample holder. High-pressure liquid nitrogen vapor is then sprayed onto the sample at high speed, freezing the sample. High-pressure freezing, similar to plunge freezing, uses cryogenic liquids to freeze the sample, but simultaneously applies a high pressure of approximately 2000 atmospheres to the sample chamber. This lowers the freezing temperature of water and suppresses the volume expansion caused by ice crystallization, thus preventing damage to the sample structure and producing higher-quality frozen biological samples.

[0006] However, insertion freezing has the following defects: since the entire sample needs to be inserted into the cryogenic liquid, it is impossible to selectively freeze specific areas of the sample during the freezing process, and it is also impossible to perform real-time microscopic observation in situ during the freezing process. Based on insertion freezing, jet freezing uses liquid nitrogen vapor instead of cryogenic liquid to improve heat transfer efficiency. High-pressure freezing is similar to the principles of the above two freezing methods. Since high pressure inhibits ice crystallization, the freezing effect is better and the sample quality is higher. However, jet freezing and high-pressure freezing also have the defects of not being able to perform real-time microscopic observation and local selective freezing. These defects limit people's further in-depth research on frozen biological samples.

[0007] A device for rapidly freezing samples is also known in the prior art. The device includes a sample container and a heating support device located on the side of the container to support the sample container. The sample container is placed on a base, and the sample is rapidly frozen by controlling the heating support device. However, because the heating support device is located on the side of the sample container, the freezing speed of the sample in this device is less than ideal.

[0008] In addition, when it comes to heating and reviving frozen biological samples, the current conventional methods have a slow heating speed and usually require the addition of auxiliary media such as DMSO to the samples to ensure that the biological samples are not destroyed during the heating process. This affects the activity of the biological samples and cannot express the true performance of biological samples such as cells in a normal environment.

[0009] In addition, the existing technology has also proposed a freezing sample preparation system that achieves visual rapid freezing of samples. The system uses a cold source, a thermal insulation layer, a heating coil, and PDMS to seal the sample chamber. By turning on the heating coil to maintain the sample room temperature and turning off the heating coil to freeze, it successfully achieved a freezing process from 25°C to a temperature close to liquid nitrogen. In the temperature range of 25°C to -21°C, a maximum of 2×10 4 °C / s freezing rate, and then as the temperature drops, the freezing rate also gradually decreases. The core of this system is a sample cavity formed by a 3-micron-thick PDMS seal. Since the cavity needs to carry a 30-micron-thick liquid layer, the bottom of the cavity needs to maintain certain mechanical properties, so the bottom wall thickness cannot be further reduced. The thermal conductivity of PDMS is only 0.15W / Km, which severely limits the further improvement of the freezing rate. In order to keep the structure of the sample from being destroyed during the freezing process at a lower freezing rate, this system adds antifreeze to the sample liquid, which affects the activity of the biological sample. At the same time, this structure cannot achieve extremely fast heating. In addition, the hardness of PDMS is relatively low, and it is easy to bend and deform during processing and operation, resulting in a poor fit between the liquid cavity and the heating coil, which also has a certain impact on the stability and thermal conductivity of the system. Summary of the Invention

[0010] In order to solve the problems in the related art, the embodiments of the present disclosure provide a freezing chip, a freezing system, a sample testing system and a method.

[0011] In a first aspect, an embodiment of the present disclosure provides a freezing chip.

[0012] Specifically, the freezing chip is in contact with a low-temperature cold source for freezing a sample, and comprises: a heating layer and a heat conducting layer arranged in sequence;

[0013] Wherein, the heating layer includes at least one temperature control unit, which is arranged on the heat conductive layer; the heat generated by the temperature control unit is transferred to the low-temperature cooling source along the thickness direction of the heat conductive layer;

[0014] It also includes: a sample placement layer, which is located on the heating layer. The surface of the sample placement layer has a local temperature control area for placing samples, and the area of ​​the area is adapted to the surface area of ​​the temperature control unit.

[0015] Optionally, when there is one temperature control unit, its surface area is less than or equal to the surface area of ​​the heat conductive layer.

[0016] Optionally, the temperature control unit is a component formed by a plurality of heating elements arranged in an array or a heating element with a predetermined surface area.

[0017] Optionally, the local temperature control area is provided with at least one closed sample holding cavity and / or open sample holding cavity for holding a sample.

[0018] Optionally, the temperature control unit further includes an auxiliary temperature control unit provided on the wall of the closed sample holding cavity and / or the open sample holding cavity.

[0019] Optionally, the freezing chip further comprises:

[0020] The heat insulation element is arranged between adjacent temperature control units.

[0021] Optionally, the thermal conductivity of the thermal insulation element is not greater than the thermal conductivity of the thermal conductive layer.

[0022] Optionally, the transverse thermal conductivity of the thermal insulation element is smaller than the transverse thermal conductivity of the thermal conductive layer.

[0023] Optionally, the freezing chip further comprises:

[0024] The chip substrate has a thermal conductivity greater than that of the thermal conductive layer and is used to support the thermal conductive layer.

[0025] Optionally, a accommodating chamber is provided in the chip substrate for accommodating a low-temperature cooling source.

[0026] Optionally, the sample placement layer, heating layer, thermal conductive layer, and chip base are an integrated structure; or the sample placement layer, heating layer, thermal conductive layer, and chip base are separately arranged in sequence.

[0027] Optionally, the cryochip is provided with an optical path channel to adapt to a microscope, a photodetector, an X-ray, a Raman spectrometer, or an infrared spectrometer.

[0028] Optionally, the cryochip is made of a light-transmitting material or has a perforated channel as the light passage channel.

[0029] Optionally, the freezing chip is made using chip micro-nano processing technology.

[0030] Optionally, the thickness of the freezing chip is controlled at 0.1-2 mm.

[0031] In a second aspect, an embodiment of the present disclosure provides a sample stage assembly comprising the freezing chip described in any one of the first aspects.

[0032] Specifically, the sample stage assembly includes:

[0033] A controller electrically connected to the temperature control unit is used to adjust the temperature of the temperature control unit.

[0034] Optionally, the sample stage assembly further comprises: a sample heat sink for accommodating the freezing chip.

[0035] In a third aspect, an embodiment of the present disclosure provides a freezing system comprising the sample stage assembly described in any one of the second aspects.

[0036] Specifically, the refrigeration system includes:

[0037] Low temperature cooling source;

[0038] A heat sink base for fixing the sample stage assembly is in contact with the low-temperature cooling source.

[0039] Optionally, the refrigeration system further comprises:

[0040] A freezing medium sealing cover plate is used to seal the low-temperature cooling source.

[0041] Optionally, the refrigeration system further comprises:

[0042] The sample cover plate has an area at least capable of sealing the opening of the heat sink base.

[0043] In a fourth aspect, an embodiment of the present disclosure provides a sample testing system, comprising the freezing system described in the third aspect.

[0044] Specifically, the sample testing system includes:

[0045] A microscopic observation device and / or detection device used in conjunction with the freezing system.

[0046] Optionally, the microscopic observation device is at least one of an upright optical microscope and an electron microscope;

[0047] The detection device is at least one of a photoelectric detector, an X-ray detector, a Raman spectrometer, and an infrared spectrometer.

[0048] In a fifth aspect, an embodiment of the present disclosure provides a method for freezing a sample using the freezing system of the third aspect.

[0049] Specifically, the method includes:

[0050] Adjusting electrical parameters of the temperature control unit to maintain the average temperature of the sample at the first temperature and to maintain a temperature gradient between the sample and the low-temperature cooling source in the sample placement layer;

[0051] detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that can be provided by the low-temperature cooling source;

[0052] Before adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at the first temperature and maintaining the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer, the method further includes:

[0053] Adjusting the temperature of the local temperature control area to the first temperature;

[0054] A sample is placed in the local temperature-controlled area.

[0055] Optionally, the first temperature is changed to the second temperature within a predetermined time period.

[0056] Optionally, the predetermined time period is within 10 ms.

[0057] Optionally, the electrical parameters of the temperature control unit are adjusted by electronic equipment.

[0058] Optionally, the first temperature is the liquidus temperature of the sample, and the second temperature enables the same sample to directly transform from liquid to amorphous solid under the same environment and continuously maintain the temperature of the amorphous solid.

[0059] Optionally, the first temperature is 0°C to 40°C, and the second temperature is below -140°C.

[0060] In a sixth aspect, an embodiment of the present disclosure provides a method for heating a sample using the freezing system of the third aspect.

[0061] Specifically, the method includes:

[0062] Adjusting the electrical parameters of the temperature control unit to a second predetermined range, and then detecting and adjusting the electrical parameters to maintain the average temperature of the sample at the first temperature; or heating the sample using an external heat source, and determining that the average temperature of the sample is at the first temperature using the temperature measurement unit;

[0063] detecting and adjusting the electrical parameters so that the average temperature of the local temperature control area reaches a second temperature;

[0064] Wherein, the first temperature is greater than the second temperature.

[0065] Optionally, the method further includes:

[0066] The electrical parameters are detected and adjusted to adjust the average temperature of the local temperature control area to a second temperature.

[0067] Optionally, the second temperature is changed to the first temperature within a predetermined time period.

[0068] Optionally, the predetermined time period is within 10 ms.

[0069] Optionally, the first temperature is the liquidus temperature of the sample, and the second temperature is the temperature at which the same sample under the same environment directly changes from liquid to amorphous solid and continuously maintains the amorphous solid.

[0070] Optionally, the first temperature is 0°C to 40°C, and the second temperature is below -140°C.

[0071] In a seventh aspect, an embodiment of the present disclosure provides a method for operating a sample using the sample testing system of the fourth aspect.

[0072] Specifically, the method includes:

[0073] Adjusting electrical parameters of the temperature control unit to maintain an average temperature of the sample at the first temperature and to maintain a temperature gradient between the sample and the low-temperature cooling source in the sample placement layer;

[0074] Detect and adjust the electrical parameters to a first predetermined range to adjust the average temperature of the sample to a second temperature, and then operate the sample at the second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the low-temperature cooling source can provide.

[0075] Optionally, the method further includes:

[0076] Adjust the electrical parameters of the temperature control unit to a second predetermined range to heat the sample or use an external heat source to heat the sample to the first temperature, then repeatedly detect and adjust the electrical parameters to the first predetermined range to maintain the average temperature of the sample at the second temperature, and then operate the sample at the second temperature.

[0077] Optionally, the method further includes:

[0078] After the steps of adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample stable at the first temperature and maintaining the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer, the sample is operated at the first temperature and the starting moment for adjusting the electrical parameters to the first predetermined range is determined. At the starting moment, the electrical parameters are detected and adjusted to the first predetermined range to maintain the average temperature of the sample at the second temperature.

[0079] Optionally, the method further includes:

[0080] After operating the sample, the sample is replaced.

[0081] Optionally, the first temperature is changed to the second temperature within a first predetermined time period.

[0082] Optionally, the electrical parameters of the temperature control unit are adjusted by electronic equipment.

[0083] Optionally, the second temperature is changed to the first temperature within a second predetermined time period.

[0084] Optionally, the second predetermined time period is within 10ms.

[0085] Optionally, the first temperature is the liquidus temperature of the sample, and the second temperature is the temperature at which the same sample under the same environment directly changes from liquid to amorphous solid and continuously maintains the amorphous solid.

[0086] Optionally, the first temperature is 0°C to 40°C, and the second temperature is below -140°C.

[0087] Optionally, the method is suitable for microscopic observation of samples.

[0088] Optionally, the method also includes adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at the first temperature and maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer, and also evaluating the thermal conductivity performance between the freezing chip and the cold source based on the electrical parameters.

[0089] The technical solutions provided by the embodiments of the present disclosure may have the following beneficial effects:

[0090] (1) The freezing chip of the embodiment of the present disclosure can selectively freeze samples by providing at least one temperature control unit. For samples that do not need to be frozen, the temperature control unit is controlled to release heat to maintain the temperature gradient between the sample and the low-temperature cooling source. For samples that need to be frozen, the electrical parameters of the temperature control unit are adjusted so that the heat of the sample is transferred to the low-temperature cooling source, thereby achieving the effect of local selective freezing.

[0091] (2) The freezing chip of the embodiment of the present disclosure can freeze large-area samples as well as multiple independent small samples. The heat of the sample is conducted along the thickness direction of the heat conductive layer, and the freezing speed can be higher than 10 5 °C / s. For cell samples, rapid freezing will not damage the cell samples, which facilitates better research on cell biological behavior.

[0092] (3) The freezing chip of the embodiment of the present disclosure is provided with an optical path channel, so that it can be adapted to a testing device for in-situ characterization of the sample, such as a microscope, an X-ray device, etc., thereby achieving simultaneous in-situ real-time testing of the sample while freezing the sample, thereby improving the sample testing efficiency.

[0093] (4) The method for operating the sample by the sample testing system of the embodiment of the present disclosure can realize the operation process of freezing the sample-operating the sample, or the cycle of freezing the sample-operating the sample-heating and reviving the sample-freezing the sample-operating the sample-heating and reviving the sample, or the operation process of operating the sample before freezing-freezing the sample-operating the sample, or the cycle of operating the sample before freezing-freezing the sample-operating the sample-freezing the sample-heating and reviving the sample-operating the sample before freezing-freezing the sample-operating the sample-heating and reviving the sample, and can also replace the sample after freezing the sample-operating the sample and repeat the above process. This technical solution limits the heat capacity of the local temperature control area by designing the thermal resistance and heat exchange efficiency of each interface between the local temperature control area, the chip substrate and the low-temperature cooling source, and obtains a heat capacity higher than 10 5 The freezing and heating speed of 100 °C / s ensures that the sample structure and function are not damaged during repeated freezing and heating. This is a major improvement for operations such as freezing, in situ observation, and heating and thawing of biological samples, and has great significance and broad application prospects.

[0094] (5) The freezing chip of the embodiment of the present disclosure is designed to be integrated with the heating layer and the sample layer. The two are directly combined, which can improve the repeatability of the freezing chip performance, ensure the stability of heat conduction, and help speed up the freezing speed.

[0095] (6) In the freezing chip of the embodiment of the present disclosure, the contact surface between the heating layer and the cold source is separable. Before freezing the sample, the contact quality at the interface can be evaluated by the heating power in the constant temperature stage to ensure the freezing speed.

[0096] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Other features, objectives and advantages of the present disclosure will become more apparent through the following detailed description of non-limiting embodiments in conjunction with the accompanying drawings. In the accompanying drawings:

[0098] Figure 1a A front view of a freezing chip according to an embodiment of the present disclosure is shown;

[0099] Figure 1b A cross-sectional view taken along line DD′ of FIG. 1 is shown;

[0100] Figure 2a-2e A schematic structural diagram of a freezing chip according to an embodiment of the present disclosure is shown;

[0101] Figure 3 A schematic structural diagram of a freezing chip according to an embodiment of the present disclosure is shown;

[0102] Figure 4 A schematic structural diagram of a sample stage assembly according to an embodiment of the present disclosure is shown;

[0103] Figure 5 A schematic structural diagram of a refrigeration system according to an embodiment of the present disclosure is shown;

[0104] Figure 6 A schematic flow chart showing a method for freezing a sample according to an embodiment of the present disclosure;

[0105] Figure 7 A schematic diagram showing the basic working principle of a temperature control unit according to an embodiment of the present disclosure;

[0106] Figure 8 A schematic flow chart showing a method for heating a sample according to an embodiment of the present disclosure;

[0107] Figure 9 A schematic flow chart showing a method for microscopically observing a sample according to an embodiment of the present disclosure;

[0108] Figure 10 Show the basis Figures 2a-2e Schematic diagram of the freezing rate of the cryochip. DETAILED DESCRIPTION

[0109] Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement them. In addition, for the sake of clarity, parts not related to the description of the exemplary embodiments are omitted in the accompanying drawings.

[0110] In the present disclosure, it should be understood that terms such as "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts, or combinations thereof disclosed in the present specification, and are not intended to exclude the possibility that one or more other features, numbers, steps, actions, components, parts, or combinations thereof exist or are added.

[0111] It should also be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present disclosure may be combined with each other. The present disclosure will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0112] In the existing technology, insertion freezing has the following defects: since the sample needs to be inserted into the cryogenic liquid as a whole, it is impossible to selectively freeze specific areas of the sample during the freezing process, and it is also impossible to perform real-time microscopic observation in situ during the freezing process. Jet freezing is based on insertion freezing, and liquid nitrogen vapor is used instead of cryogenic liquid to improve the heat transfer efficiency. High-pressure freezing is similar to the principles of the above two freezing methods. Since high pressure inhibits ice crystallization, the freezing effect is better and the sample quality is higher. However, jet freezing and high-pressure freezing also have the defects of not being able to perform real-time microscopic observation and local selective freezing. These defects limit people's further in-depth research on frozen biological samples. At the same time, there is currently no mature technology that can heat and restore frozen samples at high speed.

[0113] The present disclosure is made to at least partially solve the problems in the prior art discovered by the inventors.

[0114] The cryochip disclosed herein differs from other methods, such as insert freezing, jet freezing, and high-pressure freezing, in its principle of freezing samples. The difference lies in that the sample placed on the cryochip does not come into direct contact with the freezing medium (such as liquid nitrogen). Instead, while the freezing medium cools the sample stage (typically, the chip is placed on the sample stage, which is immersed in the freezing medium), an external resistive heating method is used to maintain the sample at a high temperature. After the resistive heating is turned off, the sample heat is quickly transferred to the low-temperature sample stage, achieving rapid freezing of the sample.

[0115] Figure 1a A front view of a freezing chip according to an embodiment of the present disclosure is shown. Figure 1b A cross-sectional view taken along the DD′ direction is shown in FIG. 1 . Figure 1b The cryogenic cooling source A shown in the figure is not part of the cryogenic chip 10. In the present disclosure, the cryogenic cooling source A is a device that provides a low-temperature environment for the cryogenic chip 10 and is in direct contact with the cryogenic chip 10. For example, when the cryogenic chip 10 is in use, it is placed on a sample heat sink, which is then fixed to a heat sink base immersed in the cryogenic cooling source. Therefore, the sample heat sink also has the temperature of the cryogenic cooling source (such as liquid nitrogen) and can be regarded as the cryogenic cooling source A. The above is an illustrative description, and the present disclosure does not limit the cryogenic cooling source A.

[0116] like Figure 1a-1bAs shown, the freezing chip 10 includes: a heating layer and a heat-conducting layer 13 arranged in sequence, wherein the heating layer includes at least one temperature control unit 12, which is arranged on the heat-conducting layer 13; the temperature control unit 12 generally generates heat locally by Joule heating (current passing through a resistor to generate heat), and the heat is distributed along the thickness direction of the heat-conducting layer 13 ( Figure 1b The heat is transferred to the low-temperature cooling source A (in the direction of the arrow shown). In a specific embodiment, the heating layer and the cooling source are separable.

[0117] When using the cryochip provided by the present disclosure, before freezing a sample, the cryochip is placed on a low-temperature cooling source A. The temperature control unit 12 maintains the sample at a first temperature, such as 20°C to 30°C. At this point, a temperature gradient is formed between the sample and the low-temperature cooling source A in the thickness direction of the thermal conductive layer 13. After freezing begins, the electrical parameters of the temperature control unit 12 are adjusted, and the heat generated by the temperature control unit is conducted along the temperature gradient, thereby achieving rapid freezing of the sample. The electrical parameters are then tested and adjusted to adjust the sample temperature to a desired second temperature. For example, if the low-temperature cooling source A can provide a low temperature of -190°C, the sample temperature can be adjusted to -140°C.

[0118] It should be noted that the second temperature is determined according to the temperature of the low-temperature cooling source A and does not need to be lower than the temperature. The present disclosure does not impose any limitation on this.

[0119] The freezing chip of the embodiment of the present disclosure can selectively freeze samples by setting at least one temperature control unit. For samples that do not need to be frozen, the temperature control unit is controlled to release heat to maintain the temperature gradient between the sample and the low-temperature cooling source. For samples that need to be frozen, the electrical parameters of the temperature control unit are adjusted to transfer the heat of the sample to the low-temperature cooling source A, thereby achieving the effect of local selective freezing.

[0120] Figure 2a-2e A schematic structural diagram of a freezing chip according to an embodiment of the present disclosure is shown.

[0121] like Figure 2a As shown, the freezing chip 10 comprises: a sample placement layer 11, a heating layer, a heat conducting layer 13 and a chip base 14 arranged in sequence. The heating layer comprises at least one temperature control unit 12 arranged on the heat conducting layer 13.

[0122] The sample placement layer 11 is located on the heating layer and is used to electrically isolate the temperature control unit 12, thereby preventing the temperature control unit 12 from being exposed to the external environment and extending the service life of the cryochip. The sample placement layer 11 is made of silicon oxide or silicon nitride.

[0123] The surface of the sample placement layer 11 has a local temperature control area N for placing samples, and the area of ​​this area is adapted to the surface area of ​​the temperature control unit 12. Specifically, the boundary range of the local temperature control area N can be marked on the sample placement layer 11, and the area enclosed by the boundary range is located directly above the temperature control unit 12 and is no larger than the surface area of ​​the temperature control unit 12. The heat generated by the temperature control unit 12 is used to heat the sample placed in the local temperature control area N, and then the electrical parameters of the temperature control unit 12 are adjusted to achieve rapid freezing of the sample. This method of freezing samples can freeze large-area samples or multiple independent small samples, and the present disclosure does not impose any restrictions on this. In some cases, the sample placement layer 11 can be omitted, and the sample can be placed directly on the temperature control unit 12.

[0124] It should be noted that the wires of the temperature control unit 12 may pass through the sample placement layer 11 and be connected to the controller outside the freezing chip 10. After the sample is placed on the heating layer, the heat generated by the temperature control unit is used to heat the sample, and the heat generated by the wire part can be ignored.

[0125] The temperature control unit 12 is arranged in the thermal conductive layer 13 by using chip micro-nano processing technology. When there is only one temperature control unit 12, its surface area is less than or equal to the surface area of ​​the thermal conductive layer 13 (see Figure 2a 、 Figure 2b As shown in FIG. 1 ), the present disclosure does not impose any restrictions on this. Furthermore, multiple temperature control units 12 may be provided, corresponding to multiple local temperature control areas N. Each local temperature control area N may be independently heated and de-heated by a corresponding temperature control unit 12, thereby independently adjusting the temperature of samples placed in different local temperature control areas N. In some cases, the temperatures of samples in several local temperature control areas N may also be adjusted simultaneously, which is not a restriction in the present disclosure.

[0126] The temperature control unit 12 is a heating element with a predetermined surface area and a thickness of generally 0.1-5 μm. The material used is generally a conductive material, such as one or more composites of metals (aluminum, copper, platinum, etc.), metal compounds (titanium nitride, indium tin oxide, etc.) or semiconductors (silicon, silicon carbide, etc.).

[0127] The thermal conductive layer 13 is made of a material with lower thermal conductivity than the chip substrate 14, such as at least one of silicon oxide, silicon nitride, and a polymer. The thickness of the thermal conductive layer 13 is typically 1 to 15 μm. The thermal conductive layer 13 is preferably made of a high thermal conductivity material, such as a metal, to increase the freezing rate.

[0128] The thermal conductivity of the chip substrate 14 is greater than that of the thermal conductive layer 13. When the freezing chip 10 contacts the low-temperature cooling source A, the temperature of the chip substrate 14 is close to the low-temperature cooling source A, and the temperature gradient is concentrated in the thickness direction of the thermal conductive layer 13, which is equivalent to reducing the conduction distance of the temperature gradient to the low-temperature cooling source A, and can provide a faster freezing speed for the frozen sample. After testing, the sample freezing speed can exceed 10 5 °C / s. The chip substrate 14 also supports the thermally conductive layer 13, providing mechanical support for the cryochip 10. The chip substrate 14, serving as the mechanical carrier for the cryochip 10, is typically 0.1-2 mm thick and is typically made of silicon (e.g., silicon wafer), silicon carbide, or a high-thermal-conductivity ceramic. In some cases, the chip substrate 14 can be omitted, allowing the thermally conductive layer 13 to directly contact the low-temperature cooling source A.

[0129] The freezing chip of the disclosed embodiment can freeze large-area samples as well as multiple independent small samples. The heat of the sample is conducted along the thickness direction of the heat-conducting layer, and the freezing speed can be higher than 10 5 °C / s, for cell samples, rapid freezing of samples will not damage the cell samples, which is convenient for better study of cell biological behavior. Figure 10 As shown, for the blank chip, the temperature drops from 300K (corresponding to the horizontal axis time point 1.4ms) to 90K (corresponding to the horizontal axis time point 2.5ms) in 1.1ms, and the freezing rate reaches about 1.9×10 5 °C / s. Similarly, the water-containing chip that freezes the sample takes only 2ms to freeze the sample temperature from 300K (corresponding to the horizontal axis time point of 1.4ms) to 90K (corresponding to the horizontal axis time point of 3.4ms), and the freezing rate reaches 1.1×10 5 In this disclosure, unless otherwise specified, a blank chip refers to a chip without a sample, and a water-containing chip refers to a chip with a liquid sample.

[0130] According to the embodiments of the present disclosure, Figure 2b As shown, a receiving chamber 141 may be provided in the chip substrate 14 for receiving a low-temperature cooling source, such as liquid nitrogen, so that the temperature of the chip substrate 14 is closer to that of the low-temperature cooling source, thereby increasing the freezing speed of the sample.

[0131] According to the embodiments of the present disclosure, Figure 2cAs shown, the temperature control unit 12 is a component formed by multiple heating elements arranged in an array to provide more freedom in coordination with the heating circuit. For example, it can be composed of multiple conductive leads connected in series or in parallel. The width of each heating element can be 2μm or less, and the spacing between adjacent heating elements can be 2μm or less. This disclosure does not impose any restrictions on this. The area on the sample placement layer 11 directly above the temperature control unit 12 is the local temperature control area N. The heat generated by the temperature control unit 12 is used to heat the sample placed in the local temperature control area N, and then the electrical parameters of the temperature control unit 12 are adjusted to achieve rapid freezing of the sample.

[0132] According to the embodiments of the present disclosure, Figure 2d As shown, the heating layer includes a plurality of temperature control units 12, which can be a component formed by a plurality of heating elements arranged in an array, or a heating element with a predetermined surface area, so as to form a plurality of local temperature control areas N on the sample placement layer 11, wherein the thickness of the thermal conductive layer 13 adapted to each local temperature control area N can be the same or different, thereby forming local temperature control areas N with different thermal conductivity properties.

[0133] In the disclosed method, for the sample area that needs to be quickly frozen, a heat conductive layer 13 with a relatively small thickness and relatively large thermal conductivity (relatively low thermal resistance) can be used to achieve a temperature of more than 10 5 °C / s high-speed freezing; for areas that do not require rapid freezing, a thermally conductive layer 13 with a larger thickness and relatively small thermal conductivity (relatively high thermal resistance) can be used to freeze at a relatively slow rate, so that a smaller heating power can be used to maintain the first temperature in the area, thereby reducing the power consumed by the temperature control unit 12.

[0134] The freezing chip of the disclosed embodiment can place a large area sample in the local temperature control area N, or freeze multiple independent small samples, and then control the temperature control unit 12 to change the temperature of each local temperature control area N, and perform sample freezing or thawing operations by adjusting the electrical parameters of the temperature control unit 12.

[0135] According to the embodiments of the present disclosure, Figure 2e As shown, the cryostat chip 10 further includes a thermal insulation element 15 disposed between adjacent temperature control units 12. The thermal insulation element 15 is made of a material having a thermal conductivity no greater than that of the thermal conductive layer 13, and may be, for example, at least one of silicon oxide, silicon nitride, and a polymer. Preferably, the transverse thermal conductivity of the thermal insulation element is less than that of the thermal conductive layer.

[0136] As required, a heat-insulating element 15 is provided to occupy or penetrate one or more functional layers such as the sample placement layer 11, the heating layer, the heat-conducting layer 13 and the chip substrate. Its function is to reduce the transverse heat conduction capacity of the sample heat between adjacent temperature control units 12 and to enhance the dominance of longitudinal heat conduction in the thickness direction of the heat-conducting layer 13. In specific settings, one way is that one end of the heat-insulating element 15 extends into the interior of the chip substrate 14, and the other end is close to the heat-conducting layer 13 or the other end extends into the interior of the sample placement layer 11; one way is that one end of the heat-insulating element 15 extends into the interior of the heat-conducting layer 13, and the other end extends into the interior of the sample placement layer 11; one way is that the heat-insulating element 15 is provided in the heat-conducting layer 13, one end of the heat-insulating element 15 is close to the gap between adjacent temperature control units 12, and the other end is close to the chip substrate 14; in the case where there is only one temperature control unit 12, multiple heat-insulating elements 15 can be provided around the temperature control unit 12. In some cases, air or vacuum can be sealed between adjacent temperature control units 12 to reduce interference between adjacent local temperature control areas N, improve temperature uniformity at the edges of the areas, and reduce the heating power required to maintain the sample temperature, thereby saving resources. The above description is merely illustrative, and the present disclosure does not limit the possible configurations of the thermal insulation element 15.

[0137] above Figure 2a-2e The specific method shown is used as a schematic illustration. The sample placement layer and the heating layer are integrated structures. Compared with the design of the prior art where there is an interface between the sample and the heating layer, the sample layer and the heating layer are integrated in the present disclosure. There is no interface between the two, which is conducive to heat transfer and the freezing rate exceeds 10 5 °C / s, which is much higher than the highest freezing rate of 2×10 4 °C / s.

[0138] In addition, due to the integrated structure of the sample layer and the heating layer, the contact surface between them is smooth, which can reduce scattering, improve light transmittance, and facilitate subsequent sample testing. At the same time, due to the integrated structure between the sample layer and the heating layer, the defects of incomplete contact between the heating layer and the sample layer in the prior art are eliminated, which can ensure the stability of thermal conductivity performance, significantly improve heat transfer efficiency, and facilitate rapid freezing. In this disclosure, the stability of thermal conductivity performance refers to the temperature-time curve of the freezing rate of the freezing chip being basically consistent with good reproducibility each time it is frozen, or the temperature-time curve of the freezing rate of freezing chips produced in different batches being basically consistent.

[0139] As another embodiment, the sample placement layer and the heating layer are integrated structures, and the thermal conductive layer and the chip base can also be separately arranged in sequence. When in use, the sample placement layer and the heating layer, the thermal conductive layer, and the chip base are stacked in sequence and fixed with an external clamp; the thermal conductive layer and the chip base layer can also be integrated with the sample placement layer and the heating layer, such as being combined into one layer using chip micro-nano processing technology.

[0140] As another embodiment, the sample placement layer and the heating layer can be separated. However, the inventors found that the separated sample placement layer will generate new thermal resistance between the layers compared to the design of the integrated structure of the sample placement layer and the heating layer, which will affect the freezing speed of the freezing chip. Its freezing effect is not as good as that of the integrated structure. However, the freezing rate of the blank chip can also reach 10 5 °C / s, the freezing rate can reach 5×10 4 °C / s. Although the separation between the sample placement layer and the heating layer affects the freezing effect, it can also meet the needs of samples with slightly lower freezing rate requirements. The independent setting of the sample placement layer allows the number of sample placement layers to be flexibly set as needed, and when a sample placement layer is damaged, it can be replaced in time.

[0141] According to an embodiment of the present disclosure, the overall thickness of the freezing chip 10 is controlled to be 0.1-2 mm.

[0142] According to an embodiment of the present disclosure, the cryochip is provided with an optical path, so that it can be adapted to test equipment for in-situ characterization of samples, such as microscopes, X-ray devices, etc., thereby achieving in-situ real-time testing of frozen samples and improving testing efficiency. Specifically, the cryochip 10 is made of a light-transmitting material or has a perforated channel as the optical path, so as to adapt to detection instruments such as upright optical microscopes, electron microscopes, photodetectors, X-rays, Raman spectrometers, infrared spectrometers, etc.

[0143] In the disclosed method, a test device is usually adapted in the area above the sample to perform in-situ characterization of the sample, such as adapting an upright optical microscope for microscopic observation. When a perforated channel is provided on the freezing chip, an inverted optical microscope can be adapted for microscopic observation. It should be noted that the heat conduction of the sample along the thickness direction of the thermal conductive layer may be affected by the perforated channel, but this change has little effect on the freezing speed of the sample. In the disclosed method, the aperture of the perforated channel can be flexibly adjusted, and the present disclosure does not impose any restrictions on this.

[0144] Those skilled in the art will appreciate that, depending on design requirements, the sample placement layer, thermal conductive layer, and chip substrate may be discontinuous, and holes or grooves may be opened therein to adjust thermal conductivity or facilitate optical observation.

[0145] Figure 3 FIG. 1 shows a schematic structural diagram of a freezing chip according to an embodiment of the present disclosure. Figure 3 As shown, the difference from Figure 1 is that the local temperature control area is provided with at least one closed sample holding cavity a and / or open sample holding cavity b for holding a sample. For other technical contents of the freezing chip of the embodiment of the present disclosure, please refer to the embodiment shown in Figure 1 and will not be repeated here.

[0146] According to an embodiment of the present disclosure, the temperature control unit 12 further includes an auxiliary temperature control unit disposed on the wall of the closed sample holding chamber a and / or the open sample holding chamber b, for reducing temperature differences between multiple samples placed within the same local temperature control area. In this embodiment, the auxiliary temperature control unit and the temperature control unit may utilize the same or identical components, or equivalent components.

[0147] Figure 4 FIG. 1 is a schematic structural diagram of a sample stage assembly according to an embodiment of the present disclosure. Figure 4 As shown, the sample stage assembly 20 includes a cryochip 10, a sample heat sink 21, and a controller 22. The sample heat sink 21 is used to accommodate the cryochip 10. The controller 22 is electrically connected to the temperature control unit 12 and is used to adjust the temperature of the temperature control unit 12. It should be noted that the sample heat sink 21 can be designed as a light-transmitting structure to accommodate microscopic observation of samples.

[0148] In the disclosed embodiment, the thermal resistance between the sample heat sink 21 and the low-temperature cooling source in the sample stage assembly 20 is very low, the heat transfer rate is very fast, and the sample heat sink 21 has a temperature close to that of the low-temperature cooling source in the equilibrium state, and can be used as the low-temperature cooling source A. It is understood that the sample heat sink 21 can also be omitted, and the cryochip 10 can be directly placed on the heat sink base 32 described below. In this case, the heat sink base 32 can be regarded as the low-temperature cooling source A, and this disclosure does not limit this.

[0149] In the disclosed embodiment, the sample stage assembly 20 also includes a control circuit board (not shown). This control circuit board can be embedded in the sample heat sink 21 or positioned around the area directly in contact with the cryochip 10, so long as it does not affect efficient heat transfer between the two. This disclosure does not limit the location of the control circuit board. The controller 22 is electrically connected to the temperature control unit 12 via the control circuit board, thereby adjusting the temperature of the temperature control unit 12.

[0150] Figure 5 FIG. 1 shows a schematic structural diagram of a refrigeration system according to an embodiment of the present disclosure. Figure 5As shown, the refrigeration system 30 includes: a sample stage assembly 20, a cryogenic cooling source 31, and a heat sink base 32. The cryogenic cooling source 31 can be liquid nitrogen, which is used to cool the heat sink base 32 and maintain it at a temperature close to liquid nitrogen. The heat sink base 32 is used to secure the sample stage assembly 20 and acts as a cooling source to keep the sample stage assembly 20 cool.

[0151] According to the embodiments of the present disclosure, when freezing a sample, the heat sink base 32 is in direct contact with the sample heat sink 21, keeping the temperature of the sample heat sink 21 close to or equal to the temperature of liquid nitrogen. The rest of the sample stage assembly 20, excluding the local temperature-controlled area N, is also frozen simultaneously. The controller 22 adjusts the electrical parameters of the temperature control unit 12, allowing the sample to be directly cooled by the surrounding temperature of the chip and the sample heat sink 21, which are close to or equal to the temperature of liquid nitrogen.

[0152] According to an embodiment of the present disclosure, the refrigeration system 30 further includes: a refrigeration medium sealing cover plate 33, which is used to seal the low-temperature cooling source and, in some cases, can also support the heat sink base 32 to be immersed in the low-temperature cooling source.

[0153] According to an embodiment of the present disclosure, the freezing system 30 further includes: a sample cover plate 34, the area of ​​which is at least capable of sealing the opening of the heat sink base 32. The length of the sample cover plate 34 shown in the figure extends to both ends of the freezing medium sealing cover plate 33 respectively. This arrangement is to ensure that no water vapor enters the low-temperature environment where the freezing chip is located, to prevent water vapor from condensing to form droplets attached to the sample, and to prevent the droplets from forming ice crystals in the low-temperature environment that affect the microscopic observation or property characterization of the sample. It can be understood that when the area of ​​the sample cover plate 34 is sufficient to cover the sample heat sink, it can usually seal the low-temperature environment where the freezing chip is located to prevent water vapor from entering. On this basis, the length of the sample cover plate 34 can be appropriately increased, and the present disclosure does not impose any restrictions on this.

[0154] In the disclosed method, the sample cover 34 may also be provided with an observation area or a detection area. This allows for microscopic observation of the sample through the observation area and / or characterization of the sample's properties using a detection device located in the detection area, while preventing moisture from entering the low-temperature environment. In some cases, a dry atmosphere may be provided for the low-temperature environment to address the problem of moisture condensation affecting sample observation or characterization. In this case, the sample cover 34 may be omitted.

[0155] The present disclosure further provides a sample testing system, including a freezing system 30 and a microscopic observation device and / or a detection device used in conjunction with the freezing system 30 .

[0156] According to an embodiment of the present disclosure, the microscopic observation device is at least one of an upright optical microscope and an electron microscope. The detection device is at least one of a photodetector, an X-ray, a Raman spectrometer, an infrared spectrometer, and other monitoring instruments.

[0157] Figure 6 Schematic diagram showing a method for freezing samples according to an embodiment of the present disclosure. Figure 6 As shown, the method utilizes the freezing system 30 to freeze the sample, including the following steps S110-S140.

[0158] In step S110, the temperature of the local temperature control area is adjusted to a first temperature;

[0159] In the disclosed method, first, the control circuit board is connected to the controller at room temperature; second, the controller is started to heat the temperature control unit to a set temperature slightly higher than room temperature (the temperature of the temperature control unit is determined by real-time measurement of the resistance value, such as 30°C), and is kept constant at this temperature (adjusted by resistance feedback). Since the distance between the temperature control unit and the sample is extremely small and the thermal resistance is extremely low, it can be approximately assumed that the sample temperature is also at the set temperature (such as 30°C). The typical resistance value range at this time is Rheater=50-100 ohm.

[0160] In step S120, placing a sample in the local temperature control area;

[0161] In step S130, the electrical parameters of the temperature control unit are adjusted to maintain the average temperature of the sample at the first temperature and to maintain the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer;

[0162] In the disclosed method, the sample stage assembly is placed on a chilled heat sink (approximately -190°C). The temperature of the cryochip begins to drop, and the controller automatically increases the current Iheater for resistive heating to maintain the average temperature of the sample within the local temperature control area N at a first temperature (e.g., 30°C). At this point, the typical current value range is Iheater = 50-100 mA, and the typical power of Rheater (Rheater × Iheater2) is approximately 0.3 W.

[0163] In step S140, the electrical parameters are detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the low-temperature cooling source can provide.

[0164] In the disclosed method, when freezing is required, a signal is sent through the controller to suddenly reduce the current Iheater to 0.1-1.0 mA. The sample temperature in the local temperature control area N will rapidly drop to the temperature of the heat sink base 32, and Rheater will also sharply decrease to about 1 / 7 of the Rheater at room temperature. During the entire cooling process, the control circuit maintains a small constant current (0.1-1.0 mA) to continuously measure the change in Rheater as a reference for temperature changes over time. After freezing is completed, the control circuit maintains a small current (0.1-1.0 mA) to maintain the average temperature of the sample at a second temperature (for example, -190 ° C), and continuously monitors the change in Rheater as a reference for the sample temperature.

[0165] In the disclosed method, the second temperature is determined according to the temperature of the low-temperature cooling source A and does not need to be lower than the temperature. Specifically, when the low-temperature cooling source A can provide a low temperature of -190°C, the sample temperature can be adjusted to the desired temperature, such as -140°C.

[0166] It should be noted that steps S110 and S120 are performed before placing the sample stage assembly into the heat sink base. During step S110, the temperature of the local temperature-controlled area can also be at room temperature, eliminating the need to activate the controller to heat the temperature-controlled unit. Furthermore, the order of executing steps S110 and S120 can be interchanged, and this disclosure does not impose any restrictions thereon.

[0167] The following is an explanation of the basic working principle of the temperature control unit:

[0168] Figure 7 Schematic diagram showing the basic working principle of the temperature control unit according to the embodiment of the present disclosure. Figure 7 As shown, the temperature control unit uses a four-terminal measurement connection: Force_H (I+), Sense_H (V+), Sense_L (V-), and Force_L (I-). A heating current, Iheater, is applied from I+ to I-. This current can reach a maximum of 50–200 mA. Simultaneously, a voltage difference, Vheater, is measured between V+ and V-. The current flowing through these terminals is very low (e.g., virtual ground), and its effect on the current flowing through the temperature control unit is negligible. The temperature control unit's resistance, Rheater, is measured in real time using Vheater / Iheater, and this value is used to evaluate the unit's average temperature. This allows for real-time monitoring of the unit's heating power, enabling real-time monitoring of thermal conductivity.

[0169] It should be noted that in the embodiments of the present disclosure, the function of localized selective freezing can be achieved by controlling the temperature control units corresponding to different local temperature control areas. The temperature control units and local temperature control areas can have a one-to-one correspondence. Of course, a single temperature control unit can also be used to adjust the temperature of multiple local temperature control areas as needed. Those skilled in the art can freely combine these methods to achieve the function of rapidly freezing samples. The present disclosure is not limited to this.

[0170] According to an embodiment of the present disclosure, the average temperature of the sample is adjusted by adjusting an electrical parameter, wherein the electrical parameter may be a current, a resistance, or a power parameter, which is not limited in the present disclosure.

[0171] In the disclosed method, a temperature control unit can be used to measure the sample temperature in real time while heating the sample. Alternatively, a temperature measurement unit can be separately provided on the cryochip, and the temperature control unit can be used to heat the sample while simultaneously measuring the sample temperature in real time. This disclosure does not impose any restrictions on this.

[0172] In the disclosed method, a resistance change curve over time can be plotted, and then the sample cooling rate can be evaluated based on the resistance change curve over time. Specifically, Rheater can be calculated by measuring Vheater while keeping the Iheater current constant. The Rheater change curve over time during the cooling process can be continuously monitored, and this curve can be used as a reference for evaluating the sample freezing rate.

[0173] According to an embodiment of the present disclosure, the first temperature is changed to the second temperature within a predetermined period of time.

[0174] In the disclosed method, the predetermined time period for reducing the first temperature to the second temperature is controlled within 10ms, for example, 1-2ms. Specifically, within 1ms, the temperature is reduced from room temperature to below -140°C, and further reduced to below -180°C within a subsequent 1-2ms.

[0175] According to an embodiment of the present disclosure, the time delay can be the delay time from when the control system sends an electrical signal to lower the first temperature to when the freezing chip receives the electrical signal and starts freezing the sample. It can be understood that when testing biological samples, it is necessary to determine the time point of freezing the biological sample in order to observe the sample at that time point or perform other tests. The time delay reflects the delay time of the freezing operation. The smaller the time delay, the more accurately the time point of freezing the sample can be controlled, so that the state of the sample after freezing is close to the state of the sample during the freezing operation, thereby better performing sample testing.

[0176] According to the embodiments of the present disclosure, by optimizing the circuit structure and control method of the temperature control unit, the time delay can be controlled to be less than 0.1 ms.

[0177] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, for example, an aqueous solution under normal pressure, for conventional cell samples, the temperature is in the range of 0-40°C, preferably 20-30°C; for special heat-resistant cells or bacteria, the temperature can be increased; under normal pressure conditions, the temperature range may also vary to ensure that the culture medium is in a liquid state and the biological sample survives normally.

[0178] According to an embodiment of the present disclosure, the second temperature is a temperature at which the same sample is directly transformed from a liquid state to an amorphous state under the same environment, and is continuously maintained in the amorphous state. For example, for water or a general aqueous solution, the temperature should be lower than -140°C. Under high or low pressure, the temperature range may change to ensure that the culture medium is frozen to a temperature at which the amorphous state is stable, thereby not destroying the sample structure.

[0179] According to an embodiment of the present disclosure, during step S310, the thermal conductivity between the freezing chip and the cold source can also be evaluated based on electrical parameters. The thermal conductivity rate between the freezing chip and the cold source is evaluated based on the resistance value of the aforementioned temperature control unit, which will not be described in detail here. It should be noted that before freezing the sample, determining whether the thermal conductivity meets the requirements based on the heating power of the temperature control unit is beneficial to improving the success rate of the experiment. Too slow thermal conductivity will result in insufficient freezing speed, while too fast thermal conductivity will increase the power required to maintain the first temperature and may also result in insufficient freezing power of the cold source, both of which are not conducive to sample preparation. By adopting a structure in which the heat-conducting layer is integrated with the sample placement layer and separated from the cold source, the contact surface between the heating layer and the cold source can be adjusted during operation, such as by re-contacting after cleaning or adjusting the contact force to optimize the thermal conductivity performance to meet the requirements. However, the structure in which the heat-conducting layer is integrated with the cold source and separated from the sample in the prior art does not have this function.

[0180] Figure 8 Schematic diagram showing a method for heating a sample according to an embodiment of the present disclosure. Figure 8 As shown, the method utilizes the freezing system 30 to heat the sample, including the following steps S210-S220.

[0181] In step S210 , electrical parameters are detected and adjusted so that the average temperature of the local temperature control area reaches a second temperature.

[0182] In the disclosed method, first, the temperature control unit is connected to the controller under low temperature (liquid nitrogen temperature); second, the control circuit is started, and the IHeater setting value is 0.1-1.0 mA (only for measuring the resistance value to evaluate the temperature, heating can be ignored), and the temperature of the temperature control unit is close to the heat sink temperature.

[0183] In step S220, the electrical parameters of the temperature control unit are adjusted to a second predetermined range, and then the electrical parameters are detected and adjusted to maintain the average temperature of the sample at a first temperature; or the sample is heated using an external heat source, and the average temperature of the sample is determined to be at a first temperature by a temperature measuring unit; wherein the first temperature is greater than the second temperature.

[0184] In the disclosed method, when the temperature of the temperature control unit is close to the temperature of the heat sink, IHeater is suddenly increased to heat the Rheater to the Rheater value corresponding to the set temperature (such as 30°C) as quickly as possible. In this process, since the initial resistance value of the Rheater at liquid nitrogen temperature is only about 1 / 7 of that at room temperature, the current of the initial heating will be very high, so as to achieve a power equivalent to 0.3W, thereby achieving the purpose of rapid heating. At the same time, during the heating process, due to the rapid increase in resistance value, Iheater needs to be quickly adjusted (reduced) to a reasonable range, so as to maintain the Rheater at the set value (such as the Rheater corresponding to 30°C). After that, the heating element is maintained stable at the set temperature (such as 30°C), and the sample can be removed as needed, or the sample can continue to be frozen.

[0185] In the disclosed method, an external heat source can be used to heat the sample by focusing the heating area to a local temperature-controlled area on the cryochip. The heating power and temperature can then be controlled by coordinating with a feedback system on the cryochip. For example, a temperature measurement unit can be provided on the cryochip to monitor the sample temperature in real time, thereby controlling the heating power of the external heat source. The external heat source can be, for example, microwaves or lasers.

[0186] It should be noted that after the sample is frozen using the freezing chip, step S210 can be omitted and step S220 can be directly performed to heat the sample.

[0187] The method for heating a sample provided by the embodiment of the present disclosure utilizes a freezing system 30 to heat the sample. For specific technical details, refer to Figure 5 The embodiments shown are not described in detail here.

[0188] According to an embodiment of the present disclosure, the second temperature is changed to the first temperature within a predetermined period of time.

[0189] According to an embodiment of the present disclosure, the predetermined time period is within 10 ms, for example, 1-2 ms.

[0190] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, for example, an aqueous solution under normal pressure, for conventional cell samples, the temperature is in the range of 0-40°C, preferably 20-30°C; for special heat-resistant cells or bacteria, the temperature can be increased; under normal pressure conditions, the temperature range may also change to ensure that the culture medium is in liquid state and the biological sample survives normally.

[0191] According to an embodiment of the present disclosure, the second temperature is a temperature at which the same sample is directly transformed from a liquid state to an amorphous state under the same environment, and is continuously maintained in the amorphous state. For example, for water or a general aqueous solution, the temperature should be lower than -140°C. Under high or low pressure, the temperature range may change to ensure that the culture medium is frozen to a temperature at which the amorphous state is stable, thereby not destroying the sample structure.

[0192] Figure 9 Schematic diagram showing a method for operating a sample according to an embodiment of the present disclosure. Figure 9 As shown, the method utilizes a sample testing system to operate a sample, including the following steps S310-S340.

[0193] In step S310, the electrical parameters of the temperature control unit are adjusted to maintain the average temperature of the sample at the first temperature and to maintain the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer;

[0194] In step S320, the electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, and then the sample is operated at the second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that can be provided by the low-temperature cooling source;

[0195] In step S330, the electrical parameters of the temperature control unit are adjusted to a second predetermined range to heat the sample, or the sample is heated to the first temperature using an external heat source, and then the electrical parameters are repeatedly detected and adjusted to the first predetermined range to maintain the average temperature of the sample at the second temperature, and then the sample is operated at the second temperature;

[0196] In step S340, after operating the sample, the sample is replaced.

[0197] It should be noted that step S340 can be performed after heating the sample to the first temperature in step S320. That is, after operating the sample once at the second temperature and then heating the sample to the first temperature, the sample can be repeatedly frozen as needed. After operating the sample a second time, the sample can be heated to the first temperature before the operation is terminated. The present disclosure does not limit the number of cycles of freezing, heating, and refreezing. It is understood that after the operation is terminated in step S320, a new sample can be replaced at the first temperature and then the new sample can be frozen again. The present disclosure does not limit this.

[0198] The method for operating samples provided in the embodiments of the present disclosure is described in detail in Figure 6 、 Figure 8 The embodiments shown are not described in detail here.

[0199] According to an embodiment of the present disclosure, the operating sample may be a microscopic observation sample or a detection signal of a test sample under monitoring instruments such as a photoelectric detector, X-ray, Raman spectrometer, infrared spectrometer, etc., and the present disclosure does not impose any restrictions on this.

[0200] According to an embodiment of the present disclosure, after the step of adjusting the electrical parameters of the temperature control unit in step S310 to maintain the average temperature of the sample at the first temperature and maintaining the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer, the method further includes:

[0201] The sample is operated at a first temperature and a start time for adjusting the electrical parameter to a first predetermined range is determined. At the start time, the electrical parameter is detected and adjusted to the first predetermined range to maintain the average temperature of the sample at a second temperature.

[0202] According to an embodiment of the present disclosure, the first temperature is changed to the second temperature within a first predetermined time period.

[0203] According to an embodiment of the present disclosure, the electrical parameters of the temperature control unit are adjusted by an electronic device, for example, by using a Keithley 2612B to adjust the electrical parameters of the temperature control unit, the time delay can be controlled within 2 ms.

[0204] According to the embodiments of the present disclosure, by optimizing the circuit structure and control method of the temperature control unit, the time delay can be controlled to be less than 0.1 ms.

[0205] According to an embodiment of the present disclosure, the second temperature is changed to the first temperature within a second predetermined time period.

[0206] According to an embodiment of the present disclosure, the second predetermined time period is within 10 ms, for example, 1-2 ms.

[0207] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, for example, an aqueous solution under normal pressure, for conventional cell samples, the temperature is in the range of 0-40°C, preferably 20-30°C; for special heat-resistant cells or bacteria, the temperature can be increased; under normal pressure conditions, the temperature range may also change to ensure that the culture medium is in liquid state and the biological sample survives normally.

[0208] According to an embodiment of the present disclosure, the second temperature is a temperature at which the same sample is directly transformed from a liquid state to an amorphous state under the same environment, and is continuously maintained in the amorphous state. For example, for water or a general aqueous solution, the temperature should be lower than -140°C. Under high or low pressure, the temperature range may change to ensure that the culture medium is frozen to a temperature at which the amorphous state is stable, thereby not destroying the sample structure.

[0209] The method for operating samples by the sample testing system of the embodiment of the present disclosure can realize the operation process of freezing sample-operating sample, or the cycle of freezing sample-operating sample-heating and reviving sample-freezing sample-operating sample-heating and reviving sample, or the operation process of operating sample before freezing-freezing sample-operating sample, or the cycle of operating sample before freezing-freezing sample-operating sample-heating and reviving sample-operating sample before freezing-freezing sample-operating sample-heating and reviving sample, and can also replace the sample after freezing sample-operating sample and repeat the above process. This technical solution limits the heat capacity of the local temperature control area by designing the thermal resistance and heat exchange efficiency of each interface between the local temperature control area, the chip substrate and the low-temperature cooling source, and obtains a heat capacity higher than 10 5 The freezing and heating speed of 0.1°C / s ensures that the sample structure and function are not damaged (or the damage is reduced) during repeated freezing and heating. This is a major improvement for operations such as freezing, in-situ observation and heating and thawing of biological samples, and has great significance and broad application prospects.

[0210] The following describes in detail how to use the sample testing system provided by the embodiment of the present disclosure to observe samples microscopically.

[0211] Method 1: Place the sample in a local temperature-controlled area - maintain it at a first temperature - freeze it to a second temperature - and then observe it under a microscope. This method is suitable for protein samples, and high-resolution microscopy is performed after freezing the sample.

[0212] Method 2: Place the sample in a local temperature-controlled area - maintain it at a first temperature - observe it under a real-time microscope - start freezing it at a specific time point - maintain it at a second temperature - observe it under a high-resolution microscope. This method is suitable for cell samples. You can first observe the sample activity in real time, freeze the sample at a specific time point of interest, such as cell division or when the cell engulfs foreign matter, and then observe it under a high-resolution microscope.

[0213] It should be noted that the microscopes used for real-time microscopic observation before and after freezing can be different to achieve observations with different resolutions. For example, a conventional upright optical microscope can be used to observe the sample in real time, while an electron microscope can be used to observe the cell structure at high resolution after freezing.

[0214] The method for microscopic observation of samples provided in the embodiment of the present disclosure is to freeze the cell sample from 20-30°C to about -170°C in less than 2ms and at a freezing speed of more than 10 5 °C / s, ensuring that the cell sample maintains a basically unchanged shape after freezing, without cracking or obvious deformation.

[0215] The above description is merely a preferred embodiment of the present disclosure and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention herein is not limited to technical solutions formed by specific combinations of the aforementioned technical features. It also encompasses other technical solutions formed by any combination of the aforementioned technical features or their equivalents, without departing from the inventive concept. For example, a technical solution formed by replacing the aforementioned features with (but not limited to) technical features with similar functions disclosed in this disclosure.

Claims

1. A freezing chip, characterized in that: The freezing chip is in contact with a low-temperature cold source and is used to freeze the sample, and includes: a heating layer and a heat-conducting layer arranged in sequence; Wherein, the heating layer includes at least one temperature control unit, which is arranged on the heat conductive layer; the heat generated by the temperature control unit is transferred to the low-temperature cooling source along the thickness direction of the heat conductive layer; It also includes: a sample placement layer, which is located on the heating layer. The surface of the sample placement layer has a local temperature control area for placing samples, and the area of ​​the area is adapted to the surface area of ​​the temperature control unit.

2. The freezing chip according to claim 1, characterized in that: When the number of the temperature control unit is one, the surface area of ​​the temperature control unit is smaller than or equal to the surface area of ​​the heat conducting layer.

3. The freezing chip according to claim 1, characterized in that: The temperature control unit is a component formed by a plurality of heating elements arranged in an array or a heating element with a predetermined surface area.

4. The freezing chip according to claim 1, characterized in that: The local temperature control area is provided with at least one closed sample receiving cavity and / or open sample receiving cavity for receiving a sample.

5. The freezing chip according to claim 4, characterized in that: The temperature control unit further includes an auxiliary temperature control unit provided on the wall of the closed sample holding cavity and / or the open sample holding cavity.

6. The freezing chip according to claim 3, characterized in that: Also includes: The heat insulation element is arranged between adjacent temperature control units.

7. The freezing chip according to claim 6, characterized in that: The thermal conductivity of the thermal insulation element is no greater than the thermal conductivity of the thermal conductive layer.

8. The freezing chip according to claim 6, characterized in that: The transverse thermal conductivity of the thermal insulation element is less than the transverse thermal conductivity of the thermally conductive layer.

9. The freezing chip according to any one of claims 1-5, 7-8, characterized in that: Also includes: The chip substrate has a thermal conductivity greater than that of the thermal conductive layer and is used to support the thermal conductive layer.

10. The freezing chip according to claim 9, characterized in that: The chip substrate is provided with a receiving chamber for receiving a low-temperature cooling source.

11. The freezing chip according to any one of claims 1-5, 7-8, and 10, characterized in that: The cryochip is provided with an optical path channel to adapt to a microscope, a photodetector, an X-ray, a Raman spectrometer, and an infrared spectrometer. 12 . The freezing chip according to claim 11 , wherein the freezing chip is made of a light-transmitting material or has a perforated channel as the light passage channel.

13. The freezing chip according to any one of claims 1-5, 7-8, 10, and 12, characterized in that: The freezing chip is made by using chip micro-nano processing technology.

14. The freezing chip according to claim 13, characterized in that: The thickness of the freezing chip is controlled at 0.1-2 mm.

15. The freezing chip according to any one of claims 1-5, 7-8, 10, 12, and 14, characterized in that: The sample placement layer and the heating layer are integrated structures or separately arranged.

16. A sample stage assembly comprising the cryochip according to any one of claims 1 to 15, characterized in that: include: A controller electrically connected to the temperature control unit is used to adjust the temperature of the temperature control unit.

17. The sample stage assembly according to claim 16, wherein: Also includes: A sample heat sink is used to accommodate the freezing chip.

18. A freezing system comprising the sample stage assembly according to claim 16 or 17, characterized in that: include: Low temperature cooling source; A heat sink base for fixing the sample stage assembly is in contact with the low-temperature cooling source.

19. The refrigeration system according to claim 18, wherein Also includes: A freezing medium sealing cover plate is used to seal the low-temperature cooling source.

20. The refrigeration system according to claim 18 or 19, characterized in that Also includes: The sample cover plate has an area at least capable of sealing the opening of the heat sink base.

21. A sample testing system comprising the freezing system according to any one of claims 18 to 20, characterized in that: include; A microscopic observation device and / or detection device used in conjunction with the freezing system.

22. The sample testing system according to claim 21, characterized in that: The microscopic observation device is at least one of an upright optical microscope and an electron microscope; The detection device is at least one of a photoelectric detector, an X-ray detector, a Raman spectrometer, and an infrared spectrometer.

23. A method for freezing a sample using the freezing system according to any one of claims 18 to 20, characterized in that: include: Adjusting electrical parameters of the temperature control unit to maintain the average temperature of the sample at the first temperature and to maintain a temperature gradient between the sample and the low-temperature cooling source in the sample placement layer; detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that can be provided by the low-temperature cooling source; Before adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at the first temperature and maintaining the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer, the method further includes: Adjusting the temperature of the local temperature control area to the first temperature; A sample is placed in the local temperature-controlled area.

24. The method according to claim 23, wherein The first temperature is changed to the second temperature within a predetermined period of time.

25. The method according to claim 24, characterized in that The predetermined time period is within 10 ms.

26. The method according to claim 23, wherein The electrical parameters of the temperature control unit are adjusted by electronic equipment.

27. The method according to claim 23, characterized in that The first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly transformed from liquid to amorphous solid under the same environment and continuously maintains the amorphous solid state.

28. The method according to claim 27, characterized in that The first temperature is 0°C to 40°C, and the second temperature is below -140°C.

29. A method for heating a sample using the freezing system according to any one of claims 18 to 20, characterized in that: include: adjusting an electrical parameter of the temperature control unit to a second predetermined range, and then detecting and adjusting the electrical parameter to maintain the average temperature of the sample at the first temperature; or heating the sample by an external heat source, and determining the average temperature of the sample at the first temperature by a temperature measuring unit; detecting and adjusting the electrical parameters so that the average temperature of the local temperature control area reaches a second temperature; Wherein, the first temperature is greater than the second temperature.

30. The method according to claim 29, wherein The second temperature is changed to the first temperature within a predetermined period of time.

31. The method according to claim 30, wherein The predetermined time period is within 10 ms.

32. The method according to claim 29, wherein The first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly transformed from liquid to amorphous solid under the same environment and continuously maintains the amorphous solid state.

33. The method according to claim 32, characterized in that The first temperature is 0°C to 40°C, and the second temperature is below -140°C.

34. A method for operating a sample using the sample testing system of claim 21, wherein: include: Adjusting electrical parameters of the temperature control unit to maintain an average temperature of the sample at the first temperature and to maintain a temperature gradient between the sample and the low-temperature cooling source in the sample placement layer; Detect and adjust the electrical parameters to a first predetermined range to adjust the average temperature of the sample to a second temperature, and then operate the sample at the second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the low-temperature cooling source can provide.

35. The method according to claim 34, wherein Also includes: Adjust the electrical parameters of the temperature control unit to a second predetermined range to heat the sample or use an external heat source to heat the sample to the first temperature, then repeatedly detect and adjust the electrical parameters to the first predetermined range to maintain the average temperature of the sample at the second temperature, and then operate the sample at the second temperature.

36. The method according to claim 34 or 35, characterized in that Also includes: After the steps of adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample stable at the first temperature and maintaining the temperature gradient between the sample and the low-temperature cooling source in the sample placement layer, the sample is operated at the first temperature and the starting moment for adjusting the electrical parameters to the first predetermined range is determined. At the starting moment, the electrical parameters are detected and adjusted to the first predetermined range to maintain the average temperature of the sample at the second temperature.

37. The method according to claim 36, wherein Also includes: After operating the sample, the sample is replaced.

38. The method according to claim 34, wherein The first temperature is changed to the second temperature within a first predetermined time period.

39. The method according to claim 34, wherein The electrical parameters of the temperature control unit are adjusted by electronic equipment.

40. The method according to claim 35, wherein The second temperature is changed to the first temperature within a second predetermined period of time.

41. The method according to claim 40, wherein The second predetermined time period is within 10 ms.

42. The method according to any one of claims 34-35, 37-41, characterized in that The first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly transformed from liquid to amorphous solid under the same environment and continuously maintains the amorphous solid state.

43. The method according to claim 42, characterized in that The first temperature is 0°C to 40°C, and the second temperature is below -140°C.

44. The method according to claim 34, wherein The method is suitable for microscopic observation of samples.

45. The method according to any one of claims 34-35, 37-41, 43-44, characterized in that: The electrical parameters of the temperature control unit are adjusted to maintain the average temperature of the sample at the first temperature, maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer, and also evaluate the thermal conductivity between the freezing chip and the cold source based on the electrical parameters.

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