Frozen chip, frozen system, sample testing system and method

Abstract

The embodiments of the present disclosure disclose a freezing chip, a freezing system, a sample testing system and a method. The freezing chip comprises a chip substrate; a sample placement layer, a surface of which is divided into not less than one local temperature control area, and the local temperature control area is used for placing a sample; and a plurality of temperature control units, which are used for adjusting the temperature of the local temperature control area. The chip substrate has a supporting surface, which is used for supporting part of the top surface or bottom surface of the sample placement layer to form a first contact surface. The area of the first contact surface does not completely overlap with the projection of the local temperature control area in the same plane. The technology can select a specific time for freezing and thawing in the process of in-situ observation and characterization of the sample, and can obtain a freezing and heating speed higher than 10 5 ℃ / s through the design of the interface thermal resistance, so as to ensure that the sample is not damaged. This is a major improvement for biological sample freezing, thawing and in-situ microscopic observation and other related operations, and has great significance and wide application prospects.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN 202011583914.7, filed on December 28, 2020, the entirety of which is incorporated by reference herein. 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

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

[0005] Current biological freezing technologies mainly include plunge freezing, jetting freezing and high pressure freezing. Plunge freezing is the most commonly used sample preparation method in the industry. Plunge freezing usually fixes the sample stage (micro-grid) loaded with biological samples at the front end of the sample rod, and controls the sample to be quickly inserted into a low-temperature liquid, such as liquid ethane, or liquid nitrogen, etc., by a machine, thereby completing the freezing of the biological sample. Jetting freezing usually sends the sample stage loaded with biological samples to a specific position in the freezing chamber through the sample rod, and then uses high-pressure liquid nitrogen vapor to jet the sample at high speed, thereby completing the freezing of the biological sample. High pressure freezing is similar in principle to plunge freezing, which uses a low-temperature liquid to freeze the sample, but at the same time, a high pressure of about 2000 atmospheres is applied in the sample chamber to reduce the freezing temperature of water and inhibit the volume expansion during ice crystallization, thereby avoiding the damage of ice crystallization to the structure of the biological sample, and preparing a high-quality frozen biological sample.

[0006] However, plunge freezing has the following defects: since the sample needs to be inserted into a low-temperature liquid as a whole, it is not possible to selectively freeze specific areas of the sample during the freezing process, and real-time microscopic observation cannot be performed in situ during the freezing process. Jetting freezing uses liquid nitrogen vapor instead of low-temperature liquid based on plunge freezing, which improves the heat transfer efficiency. High pressure freezing is similar in principle to the above two freezing methods, and the freezing effect is better and the sample quality is higher due to the inhibition of ice crystallization by high pressure. However, jetting 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 further in-depth research on frozen biological samples.

[0007] A device for rapidly freezing a sample is also proposed in the prior art, which comprises a sample container and a heating support device for supporting the sample container on the side of the container. The sample container is placed on a base, and the rapid freezing of the sample is achieved by controlling the switch of the heating support device. Since the sample is arranged in a closed sample carrying structure, the wall of the sample carrying device separates the sample from the heating support device, which produces additional thermal resistance and thus results in an unsatisfactory freezing speed of the frozen sample. In addition, in terms of heating the frozen biological sample, the current conventional method has a slow heating speed, and usually needs to add auxiliary media such as DMSO in the sample to ensure that the biological sample is not damaged during the heating process, which has an impact on the activity of the biological sample and cannot express the real performance of the biological sample such as cells in the normal environment. SUMMARY

[0008] 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.

[0009] In a first aspect, the embodiments of the present disclosure provide a freezing chip.

[0010] Specifically, the freezing chip is in contact with a low-temperature cold source for freezing a sample, and the freezing chip comprises: a sample placement layer, a surface of which is divided into at least one local temperature control region for placing the sample; a plurality of temperature control units for adjusting the temperature of the local temperature control region; a chip substrate supporting the top surface or the bottom surface of the sample placement layer to form a first contact surface; and the projection of the first contact surface and the local temperature control region on the same plane does not overlap or partially overlaps.

[0011] Optionally, the chip substrate is supported on a peripheral region outside a central region of the sample placement layer, and the central region is divided into at least one local temperature control region; or the chip substrate is supported on the central region of the sample placement layer, and the peripheral region outside the central region is divided into at least one local temperature control region; or the chip substrate is supported at a spaced position of the local temperature control region.

[0012] Optionally, when the chip substrate supports the top surface of the sample placement layer to form the first contact surface, the chip substrate further has a second contact surface for contacting the low-temperature cold source; and the first contact surface and the second contact surface are located on the same side of the chip substrate.

[0013] Optionally, the temperature control unit and the sample placement layer are an integrated structure.

[0014] Optionally, the temperature control unit is arranged on the sample placement layer by using a chip micro-nano processing technology, and the local temperature control region is divided by using the temperature control unit.

[0015] Optionally, the sample placement layer is a heat-conductive layer; and the temperature control unit is arranged on the heat-conductive layer to divide the local temperature control region on the heat-conductive layer; or

[0016] The sample placement layer comprises a heat-conductive layer and a first isolation layer fabricated on the heat-conductive layer by a chip micro-nano processing technology; and the temperature control unit is arranged on the first isolation layer to divide the local temperature control region on the first isolation layer; or

[0017] The sample placement layer comprises a heat-conductive layer, a first isolation layer fabricated on the heat-conductive layer by a chip micro-nano processing technology, and a second isolation layer fabricated on the first isolation layer by a chip micro-nano processing technology; and the temperature control unit is arranged on the first isolation layer to divide the local temperature control region on the second isolation layer; or

[0018] The sample placement layer comprises a third isolation layer, a heat-conductive layer fabricated on the third isolation layer by a chip micro-nano processing technology, a first isolation layer fabricated on the heat-conductive layer by a chip micro-nano processing technology, and a second isolation layer fabricated on the first isolation layer by a chip micro-nano processing technology; and the temperature control unit is arranged on the first isolation layer to divide the local temperature control region on the second isolation layer; or

[0019] The sample placement layer comprises a third isolation layer, a first isolation layer fabricated on the third isolation layer by a chip micro-nano processing technology, a heat-conductive layer fabricated on the first isolation layer by a chip micro-nano processing technology, and a second isolation layer fabricated on the heat-conductive layer by a chip micro-nano processing technology; and the temperature control unit is arranged on the third isolation layer to divide the local temperature control region on the second isolation layer.

[0020] Optionally, the sample placement layer comprises at least one sample layer, a heating layer, a fourth isolation layer, a heat-conductive layer, and a fifth isolation layer arranged separately; the surface of the sample layer is divided into at least one local temperature control region; and the temperature control unit is arranged on the heating layer.

[0021] Optionally, the thickness of the part of the heat-conductive layer close to the temperature control unit and the end part of the heat-conductive layer is greater than the thickness of the part of the heat-conductive layer between them; and / or the part of the heat-conductive layer between the part close to the temperature control unit and the end part of the heat-conductive layer is arranged in a patterned structure.

[0022] Optionally, the local temperature control region is provided with at least one closed sample accommodation cavity and / or an open sample accommodation cavity for accommodating a sample.

[0023] Optionally, the temperature control unit further comprises an auxiliary temperature control unit arranged on the wall of the closed sample accommodating cavity and / or the open sample accommodating cavity.

[0024] Optionally, the sample placement layer is provided with a light passage channel to adapt to a microscope, a photodetector, X-ray, a Raman spectrometer, an infrared spectrometer.

[0025] Optionally, the freezing chip is made of a light-transmitting material or has a perforated channel as the light passage channel.

[0026] Optionally, the freezing chip is made by a chip micro-nano processing process.

[0027] Optionally, the thickness of the freezing chip is controlled to be 0.1-2 mm.

[0028] In a second aspect, the embodiments of the present disclosure provide a sample stage assembly comprising the freezing chip of any one of the first aspect. Specifically, the sample stage assembly comprises a controller electrically connected with the temperature control unit, for adjusting the temperature of the temperature control unit.

[0029] Optionally, the sample stage assembly further comprises a sample heat sink for accommodating the freezing chip.

[0030] In a third aspect, the embodiments of the present disclosure provide a freezing system comprising the sample stage assembly of any one of the second aspect. Specifically, the freezing system comprises a low-temperature cold source; a heat sink base for fixing the sample stage assembly, in contact with the low-temperature cold source.

[0031] Optionally, the freezing system further comprises:

[0032] a freezing medium sealing cover plate for sealing the low-temperature cold source.

[0033] Optionally, the freezing system further comprises:

[0034] a sample cover plate with an area at least capable of sealing the opening of the heat sink base.

[0035] In a fourth aspect, the embodiments of the present disclosure provide a sample testing system comprising the freezing system of the third aspect. Specifically, the sample testing system comprises:

[0036] a microscopic observation device and / or a detection device used in conjunction with the freezing system.

[0037] Optionally, the microscopic observation device is at least one of an upright optical microscope, an inverted optical microscope, and an electron microscope; and the detection device is at least one of a photodetector, X-ray, a Raman spectrometer, and an infrared spectrometer.

[0038] In a fifth aspect, the embodiments of the present disclosure provide a method for freezing a sample by using the freezing system of the third aspect. Specifically, the method comprises: adjusting an electrical parameter of a temperature control unit to maintain an average temperature of the sample at a first temperature, and maintaining a temperature gradient between the sample and a low-temperature heat source in a 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 a required temperature value is determined within a lowest temperature range that can be provided by the low-temperature heat source.

[0039] Optionally, before the adjusting of the electrical parameter 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 heat source in the sample placement layer, the method further comprises: adjusting a temperature of a local temperature control region to the first temperature; and placing the sample in the local temperature control region.

[0040] Optionally, the first temperature is changed to the second temperature within a predetermined time period.

[0041] Optionally, the electrical parameter of the temperature control unit is adjusted by an electronic device.

[0042] Optionally, the first temperature is a liquid temperature of the sample, and the second temperature is a temperature at which the same sample directly changes from a liquid state to an amorphous solid state in the same environment and continuously maintains the amorphous solid state.

[0043] Optionally, the first temperature is 0-40°C, and the second temperature is lower than -140°C.

[0044] In a sixth aspect, the embodiments of the present disclosure provide a method for heating a sample by using the freezing system of the third aspect. Specifically, the method comprises: adjusting an electrical parameter of a temperature control unit to a second predetermined range, and then detecting and adjusting the electrical parameter to maintain an average temperature of the sample at a first temperature; or heating the sample by using an external heat source, and determining the average temperature of the sample at the first temperature by using a temperature measurement unit; wherein the first temperature is greater than the second temperature.

[0045] Optionally, the method further comprises:

[0046] detecting and adjusting the electrical parameter to make the average temperature of the local temperature control region to the second temperature.

[0047] Optionally, the second temperature is increased to the first temperature within a predetermined time period.

[0048] Optionally, the predetermined time period is within 10 ms.

[0049] Optionally, the first temperature is a liquid temperature of the sample, and the second temperature is a temperature at which the same sample is directly transformed from the liquid state to the amorphous solid state under the same environment and continuously maintained in the amorphous solid state.

[0050] Optionally, the first temperature is 0-40℃, and the second temperature is lower than -140℃.

[0051] In a seventh aspect, the embodiments of the present disclosure provide a method for operating a sample by using the sample testing system of the fourth aspect.

[0052] Specifically, the method comprises: adjusting an electrical parameter of the temperature control unit to maintain an average temperature of the sample at a first temperature and maintain a temperature gradient of the sample to the low-temperature cold source in a 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, and then operating the sample at the second temperature, wherein the second temperature is lower than the first temperature, and a required temperature value is determined within a lowest temperature range that can be provided by the low-temperature cold source.

[0053] Optionally, the method further comprises: adjusting the electrical parameter of the temperature control unit to a second predetermined range to heat the sample to the first temperature or using an external heat source to heat the sample to the first temperature, and then repeatedly detecting and adjusting the electrical parameter to the first predetermined range to maintain the average temperature of the sample at the second temperature, and then operating the sample at the second temperature.

[0054] Optionally, the method further comprises: after the step of adjusting the electrical parameter of the temperature control unit to maintain the average temperature of the sample at the first temperature and maintain the temperature gradient of the sample to the low-temperature cold source in the sample placement layer, operating the sample at the first temperature and determining a starting time of adjusting the electrical parameter to the first predetermined range, at the starting time, detecting and adjusting the electrical parameter to the first predetermined range to maintain the average temperature of the sample at the second temperature.

[0055] Optionally, the method further comprises: after operating the sample, replacing the sample.

[0056] Optionally, the first temperature is changed to the second temperature within a first predetermined time period.

[0057] Optionally, the electrical parameter of the temperature control unit is adjusted by an electronic device.

[0058] Optionally, the second temperature is changed to the third temperature within a second predetermined time period.

[0059] Optionally, the second predetermined time period is within 10 ms.

[0060] Optionally, the first temperature is a liquid temperature of the sample, and the second temperature is a temperature at which the same sample is directly converted from a liquid state to an amorphous solid state and continuously maintained in the amorphous solid state under the same environment.

[0061] Optionally, the first temperature is 0-40℃, and the second temperature is lower than -140℃.

[0062] Optionally, the method is applicable to microscopic observation of the sample.

[0063] The technical solution provided by the embodiments of the present disclosure can include the following beneficial effects:

[0064] (1) The freezing chip of the embodiments of the present disclosure can selectively freeze the sample by setting at least one local temperature control area, adjusting the temperature of the local temperature control area by the temperature control unit, for the sample that does not need to be frozen, controlling the temperature control unit to release heat to maintain the temperature gradient of the sample and the low-temperature heat source, and for the sample that needs to be frozen, adjusting the electrical parameters of the temperature control unit to make the sample heat conduct to the low-temperature heat source, thereby realizing the effect of local selective freezing.

[0065] (2) The freezing chip of the embodiments of the present disclosure, the temperature control unit is integrally arranged with the sample placement layer, when the freezing chip is in contact with the low-temperature heat source, a temperature gradient of the sample and the low-temperature heat source is formed in the sample placement layer, by adjusting the electrical parameters of the temperature control unit, the sample heat can be quickly conducted along the direction of the temperature gradient, thereby realizing the rapid freezing of the sample, and providing low-temperature sample preparation for other testing devices, such as microscopes, X-ray devices, etc.

[0066] (3) The freezing chip of the embodiments of the present disclosure, by designing the structure of the heat conduction layer, the temperature gradient is limited to the part of the heat conduction layer between the part of the heat conduction layer close to the temperature control unit and the end part of the heat conduction layer, while ensuring the heat transfer speed, the heat capacity of the frozen part is reduced, so that the freezing speed is higher than 10 5 ℃ / s, for cell samples, the rapid freezing of the sample will not damage the cell sample, and it is convenient to better study the biological behavior of the cell.

[0067] (4) The freezing chip of the embodiments of the present disclosure, the sample placement layer has a light passage channel, so that the testing device can be adapted to in-situ characterization of the sample, such as microscopes, X-ray devices, etc., thereby realizing the simultaneous, in-situ real-time testing of the frozen sample, and improving the sample testing efficiency.

[0068] (5) The sample testing system of the embodiments of the present disclosure is used to operate a sample, and by adjusting the parameters of the temperature control unit, the operation process of freezing the sample and operating the sample can be realized, or the cycle of the above process of freezing the sample, operating the sample, heating the sample, freezing the sample, operating the sample, and heating the sample, or the operation process of pre-freezing operation of the sample, freezing the sample, and operating the sample, or the cycle of the above process of pre-freezing operation of the sample, freezing the sample, operating the sample, heating the sample, pre-freezing operation of the sample, freezing the sample, operating the sample, and heating the sample, or the above process can be repeated after replacing the sample after freezing the sample and operating the sample. The technical scheme limits the heat capacity of the local temperature control area by designing the thermal resistance and heat exchange efficiency of the interface between the local temperature control area, the chip substrate, and the low-temperature cold source, and obtains a freezing and heating speed higher than 10 5 ℃ / s, which ensures that the structure and function of the sample are not damaged in the repeated freezing and heating process. This is a major improvement for biological sample freezing, in-situ observation, and heating and thawing operations, and has great significance and wide application prospects.

[0069] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0070] Other features, objects, and advantages of the present disclosure will become more apparent from the following detailed description of the non-limiting embodiments in conjunction with the accompanying drawings. In the drawings:

[0071] Figure 1a A front view of a freezing chip according to an embodiment of the present disclosure is shown;

[0072] Figure 1b A cross-sectional view in the direction of DD' of FIG. 1 is shown;

[0073] Figure 1c A cross-sectional view of a freezing chip according to another embodiment of the present disclosure is shown;

[0074] Figure 1d A cross-sectional view of a freezing chip according to another embodiment of the present disclosure is shown;

[0075] Figures 2a-2e A structural schematic diagram of a sample placement layer according to an embodiment of the present disclosure is shown;

[0076] Figure 3 A schematic diagram of a temperature gradient in a sample placement layer according to an embodiment of the present disclosure is shown;

[0077] Figure 4 A structural schematic diagram of a freezing chip for placing a sample according to an embodiment of the present disclosure is shown;

[0078] Figure 5A schematic diagram of the sample stage assembly according to an embodiment of the present disclosure is shown;

[0079] Figure 6 A schematic diagram of the structure of a refrigeration system according to an embodiment of the present disclosure is shown;

[0080] Figure 7 A schematic flowchart of a method for freezing samples according to an embodiment of the present disclosure is shown;

[0081] Figure 8 This diagram illustrates the basic operating principle of the temperature control unit according to an embodiment of the present disclosure.

[0082] Figure 9 A schematic flowchart of a method for heating a sample according to an embodiment of the present disclosure is shown;

[0083] Figure 10 A schematic flowchart illustrating a method for microscopic observation of a sample according to an embodiment of the present disclosure is shown.

[0084] Figure 11 A schematic diagram showing the chip and on-chip cell samples before and after freezing;

[0085] Figure 12 Showing according to Figures 2a-2e A schematic diagram of the freezing rate of the cryogenic chip. Detailed Implementation

[0086] In the following, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings to enable those skilled in the art to readily implement them. Furthermore, for clarity, portions unrelated to the description of exemplary embodiments have been omitted from the drawings.

[0087] In this disclosure, it should be understood that terms such as “comprising” or “having” are intended to indicate the presence of features, figures, steps, behaviors, components, parts or combinations thereof disclosed in this specification, and are not intended to exclude the possibility of the presence or addition of one or more other features, figures, steps, behaviors, components, parts or combinations thereof.

[0088] It should also be noted that, unless otherwise specified, the embodiments and features described in this disclosure can be combined with each other. This disclosure will now be described in detail with reference to the accompanying drawings and embodiments.

[0089] The prior art has the following defects: in the immersion freezing, the sample is inserted into the low-temperature liquid as a whole, so that the sample cannot be selectively frozen in a specific area during the freezing process, and the sample cannot be observed in situ in real time during the freezing process. The spray freezing uses liquid nitrogen vapor instead of low-temperature liquid to replace the immersion freezing, thereby improving the heat transfer efficiency. The high-pressure freezing has a similar principle to the above two freezing methods, and the freezing effect is better and the sample quality is higher due to the suppression of ice crystallization by high pressure. However, the spray freezing and the high-pressure freezing also have the defects of being unable to observe in real time and being unable to freeze in a specific area. These defects limit the further in-depth study of the frozen biological sample. Meanwhile, there is no mature technology for rapidly heating and recovering the frozen sample.

[0090] The present disclosure is proposed to at least partially solve the problems in the prior art found by the inventors.

[0091] The freezing chip provided by the present disclosure is different from the immersion freezing, the spray freezing and the high-pressure freezing in the principle of freezing the sample. The difference lies in that the sample placed on the freezing chip is not in direct contact with the freezing medium (such as liquid nitrogen), but is kept at a higher temperature by using an external resistance heating method while the freezing medium cools the sample stage (usually the chip is placed on the sample stage, and the sample stage is immersed in the freezing medium). After the resistance heating is turned off, the heat of the sample is quickly transferred to the low-temperature sample stage, thereby realizing the rapid freezing of the sample.

[0092] Figure 1a A front view of a freezing chip according to an embodiment of the present disclosure is shown. Figures 1b-1d The low-temperature cold source A shown in the middle is not part of the freezing chip 10. In the present disclosure, the low-temperature cold source A is a device that provides a low-temperature environment for the freezing chip 10 and is in direct contact with the freezing chip 10. For example, when the freezing chip 10 is used, the sample heat sink is placed on the sample heat sink, and then the sample heat sink is fixed to the heat sink base immersed in the low-temperature cold source. Therefore, the sample heat sink also has the temperature of the low-temperature cold source (such as liquid nitrogen) and can be regarded as a low-temperature cold source A. The above is an illustrative description, and the present disclosure does not limit the low-temperature cold source A.

[0093] As shown in FIG. 1, Figures 1a-1d The freezing chip 10 includes a chip substrate 11, a sample placement layer 12 and a plurality of temperature control units 13. The chip substrate 11 is in contact with the top surface or the bottom surface of the sample placement layer 12, forming a first contact surface 14. The surface of the sample placement layer 12 is divided into at least one local temperature control area N for placing the sample. The temperature control unit 13 usually generates heat in a local area by Joule heating (electric current passing through a resistor generates heat) to adjust the temperature of the local temperature control area N. The projection of the area P of the first contact surface 14 on the same plane as the local temperature control area N does not overlap or partially overlaps.

[0094] According to an embodiment of the present disclosure, the top surface of the sample placement layer 12 is used for placing samples, and the bottom surface of the chip substrate 11 generally supports the sample placement layer 12 to form the first contact surface 14 (as shown in Figure 1b The chip substrate 11 can also support the top surface of the sample placement layer 12 to form the first contact surface (as shown in Figure 1c The chip substrate 11 supports the top surface of the sample placement layer 12, and Figure 1d The chip substrate 11 supports the top surface of the sample placement layer 12, and Figure 1c The chip substrate 11 supports the top surface of the sample placement layer 12 to form the first contact surface, and the chip substrate 11 also has a second contact surface for contacting the low-temperature cold source A; wherein the first contact surface and the second contact surface are located on the same side of the chip substrate. According to an embodiment of the present disclosure, the sample can be in direct contact with the sample placement layer 12, which can avoid additional thermal resistance and improve the freezing rate.

[0095] The above Figures 1b-1d The above

[0096] According to an embodiment of the present disclosure, the same plane can be the plane where the sample placement layer 12 is located. As shown in Figure 1b The region P of the first contact surface 14 does not overlap the local temperature control region N, and the sample heat in the local temperature control region N is conducted laterally from the local temperature control region N to the region P, and then conducted along the chip substrate 11 to the low-temperature cold source A. Figure 1c The region P of the first contact surface 14 does not overlap the local temperature control region N, and the sample heat in the local temperature control region N is conducted laterally from the local temperature control region N to the region P, and then conducted along the chip substrate 11 to the low-temperature cold source A. Figure 1b The region P of the first contact surface 14 does not overlap the local temperature control region N, and the sample heat in the local temperature control region N is conducted laterally from the local temperature control region N to the region P, and then conducted along the chip substrate 11 to the low-temperature cold source A.

[0097] It should be noted that the wires of the temperature control unit 13 can pass through the sample placement layer 12 to connect with the controller outside the freezing chip 10, and the heat generated by the wires during adjustment of the temperature of the sample in the local temperature control region N can be ignored.

[0098] Figures 1b-1dThe sample heat can be conducted to the low-temperature cold source A in the transverse direction and the longitudinal direction, so as to freeze the sample. Moreover, the center region of the chip substrate 11 is hollow, and can be adapted to a testing device for in-situ characterization of the sample, such as a microscope, an X-ray device, and the like, which is not limited in the present disclosure.

[0099] In use of the freezing chip provided by the present disclosure, before freezing the sample, the freezing chip is placed on the low-temperature cold source A, and the temperature control unit 13 maintains the sample at a first temperature, such as 20-30°C. At this time, a temperature gradient of the sample and the low-temperature cold source A is formed in the sample placement layer. After the freezing starts, the electrical parameters of the temperature control unit 13 are adjusted, and the sample heat in the local temperature control region N is conducted along the temperature gradient direction, so as to realize the rapid freezing of the sample. Then, the electrical parameters are detected to adjust the sample temperature to a second temperature, such as -140°C when the low-temperature cold source A can provide a low temperature of -190°C.

[0100] It should be noted that the second temperature is determined according to the temperature of the low-temperature cold source A, and is not lower than the temperature, which is not limited in the present disclosure.

[0101] The freezing chip of the embodiment of the present disclosure can selectively freeze the sample by setting at least one local temperature control region, adjusting the temperature of the local temperature control region by the temperature control unit. For the sample that does not need to be frozen, the heat of the temperature control unit is released to maintain the temperature gradient of the sample and the low-temperature cold source. For the sample that needs to be frozen, the electrical parameters of the temperature control unit are adjusted to conduct the sample heat to the low-temperature cold source, so as to realize the effect of local selective freezing.

[0102] According to the embodiment of the present disclosure, the chip substrate 11 is supported on the peripheral region outside the center region of the sample placement layer 12; and the center region of the sample placement layer 12 is divided into at least one local temperature control region. For example, the chip substrate 11 is a surrounding structure adapted to the periphery outside the center region of the sample placement layer 12, and supports the top surface or the bottom surface of the sample placement layer 12; or the chip substrate 11 is an independent support block supported on one side or both sides of the center region, and the like; wherein the center region is divided into at least one local temperature control region N. The upper or lower side of the center region can be adapted to a testing device for in-situ characterization of the sample, such as a microscope, an X-ray device, and the like.

[0103] As another implementation, the chip substrate 11 is supported on the center region of the sample placement layer 12; and the peripheral region outside the center region is divided into at least one local temperature control region N. For example, the freezing chip is T-shaped, and the sample placement layer 12 is horizontally arranged. The region supported by the chip substrate 11 is not used to divide the local temperature control region N, but a plurality of local temperature control regions N are divided around the support region.

[0104] In some cases, the chip substrate 11 can also be supported at intervals in the local temperature control region N. For example, the chip substrate 11 is at least two separate support blocks, respectively used to support the sample placement layer 12, and the local temperature control region N can be divided in the area between the support blocks, and the peripheral area outside the support blocks.

[0105] According to an embodiment of the present disclosure, the freezing chip 10 is made by chip micro-nano processing technology, such as thin film deposition technology, dry or wet etching technology, photolithography technology, etc. in the chip field, which is not described herein.

[0106] According to an embodiment of the present disclosure, the overall thickness of the freezing chip 10 is controlled to be 0.1-2mm.

[0107] According to an embodiment of the present disclosure, the sample placement layer 12 is provided with a light passage channel, so that the test device can be adapted to in-situ characterization of the sample, such as a microscope, an X-ray device, etc., so as to realize simultaneous, in-situ real-time testing of the frozen sample, and improve the sample testing efficiency. Specifically, the freezing chip is made of a light-transmitting material or has a perforated channel as the light passage channel, to adapt to upright optical microscopes, inverted optical microscopes, electron microscopes, photoelectric detectors, X-rays, Raman spectrometers, infrared spectrometers, etc. monitoring instruments.

[0108] According to an embodiment of the present disclosure, the chip substrate 11 is used as a mechanical carrier part of the freezing chip 10, and the thickness of the chip substrate 11 is usually 0.1-2mm, and the material used is usually silicon (such as a silicon wafer) or silicon carbide.

[0109] According to an embodiment of the present disclosure, the temperature control unit 13 is arranged in the sample placement layer 12 by chip micro-nano processing technology, and the local temperature control region N is divided by the temperature control unit 13. Each local temperature control region N can be independently controlled by the corresponding temperature control unit 13 to heat and stop heating, so as to independently adjust the temperature of the sample placed in different local temperature control regions N, and in some cases, the temperature of the samples in several local temperature control regions N can also be adjusted together, which is not limited by the present disclosure.

[0110] According to an embodiment of the present disclosure, the thickness of the temperature control unit 13 is usually 0.1-5um, and the material used is usually a conductive material, such as a metal (aluminum, copper, platinum, etc.), a metal compound (titanium nitride, indium tin oxide, etc.) or a semiconductor (silicon, silicon carbide, etc.).

[0111] Figures 2a-2e A structural schematic diagram of a sample placement layer according to an embodiment of the present disclosure is shown. As shown in FIG. 2, the sample placement layer 12 is provided with a plurality of local temperature control regions N, and each local temperature control region N is independently controlled by the temperature control unit 13. Figures 2a-2eAs shown, the sample placement layer 12 includes: a thermally conductive layer 121, a first isolation layer 122, a second isolation layer 123, and a third isolation layer 124. The thermally conductive layer 121 is used to laterally conduct heat from the sample to the low-temperature cold source A. The first isolation layer 122 isolates the thermally conductive layer 121 from the temperature control unit 13. The second isolation layer 123 isolates the temperature control unit 13 from its external environment, providing insulation and protection. The third isolation layer 124 isolates the chip substrate 11 from the thermally conductive layer 121. The first isolation layer 122, second isolation layer 123, and third isolation layer 124 can be omitted depending on the situation.

[0112] The thermally conductive layer 121 can be made of metal (such as aluminum, copper, platinum, etc.), thermally conductive ceramic (alumina, aluminum nitride, etc.), or other thermally conductive materials (such as silicon, silicon carbide, silicon nitride, etc.). The thickness of the thermally conductive layer 121 is typically 0.1-5 μm.

[0113] According to embodiments of this disclosure, the temperature control unit 13 and the sample placement layer 12 are an integrated structure.

[0114] like Figure 2a As shown, the sample placement layer 12 consists only of a thermally conductive layer 121; the temperature control unit 13 is disposed on the thermally conductive layer 121 to divide the local temperature control region N on the thermally conductive layer 121. In this embodiment, the sample placement layer 12 is composed only of a thermally conductive layer, resulting in high power consumption for maintaining the sample temperature, but it has a high freezing rate; the freezing rate of the cryogenic chip can reach 10. 5 -10 6 ℃ / s.

[0115] like Figure 2b As shown, the sample placement layer 12 includes: a thermally conductive layer 121 and a first isolation layer 122 fabricated on the thermally conductive layer 121 using chip micro / nano fabrication technology; wherein, the temperature control unit 13 is disposed on the first isolation layer 122 to divide the local temperature control area on the first isolation layer 122. In this embodiment, the freezing speed of the freezing chip is compared to... Figure 2a Even at a relatively small size, it can still reach 10. 5 -10 6 ℃ / s.

[0116] like Figure 2cAs shown, the sample placement layer 12 includes: a thermally conductive layer 121, a first isolation layer 122 fabricated on the thermally conductive layer 121 using chip micromachining technology, and a second isolation layer 123 fabricated on the first isolation layer 122 using chip micromachining technology; wherein, the temperature control unit 13 is disposed on the first isolation layer 122 to divide the local temperature control area N on the second isolation layer 123. In this embodiment, the second isolation layer 123 is disposed on the first isolation layer 122, which avoids the temperature control unit 13 from being exposed to the external environment, thereby extending the service life of the cryogenic chip. After testing, the freezing rate of the cryogenic chip can still reach 10. 5 -10 6 ℃ / s.

[0117] like Figure 2d As shown, the sample placement layer 12 includes: a third isolation layer 124, a thermally conductive layer 121 fabricated on the third isolation layer 124 using chip micro / nano fabrication technology, a first isolation layer 122 fabricated on the thermally conductive layer 121 using chip micro / nano fabrication technology, and a second isolation layer 123 fabricated on the first isolation layer 122 using chip micro / nano fabrication technology; wherein, the temperature control unit 13 is disposed on the first isolation layer 122 to divide the local temperature control region N on the second isolation layer 123. In this embodiment, a third isolation layer 124 is disposed under the thermally conductive layer 121. Considering that the thermally conductive layer 121 is usually made of metal, and based on the convenience of the processing technology, a third isolation layer 124 can be disposed between the thermally conductive layer 121 and the chip substrate 11, thereby meeting the process requirements. After testing, the freezing speed of the frozen chip can still reach 10. 5 ℃ / s.

[0118] like Figure 2e As shown, the sample placement layer 12 includes: a third isolation layer 124, a first isolation layer 122 fabricated on the third isolation layer 124 using chip micro-nano fabrication technology, a thermally conductive layer 121 fabricated on the first isolation layer 122 using chip micro-nano fabrication technology, and a second isolation layer 123 fabricated on the thermally conductive layer 121 using chip micro-nano fabrication technology; wherein, the temperature control unit 13 is disposed on the third isolation layer 124 to divide the local temperature control area on the second isolation layer 123. In this embodiment, with Figure 2d Unlike the previous implementation, the temperature control unit 13 is located below the heat-conducting layer 121, closer to the chip substrate 11 and the low-temperature cold source A, thus resulting in higher power consumption. However, testing showed that the freezing speed of the freezing chip could still reach 10. 5 ℃ / s.

[0119] Specifically, such as Figure 12As 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 2.6ms) in 1.2ms, and the freezing rate reaches about 1.8x10 5 ℃ / s. Similarly, the water-containing chip freezes the sample temperature from 300K (corresponding to the horizontal axis time point 1.4ms) to 90K (corresponding to the horizontal axis time point 3.6ms) in only 2.2ms, and the freezing rate reaches 1.0x10 5 ℃ / s. In the present disclosure, unless otherwise specified, the blank chip refers to a chip without loading the sample, and the water-containing chip refers to a chip loading the liquid sample.

[0120] Figures 2a-2e In the present disclosure, the material of the heat-conducting layer 121 is preferably a high-thermal-conductivity material, such as a metal material, to improve the freezing speed.

[0121] The above-mentioned specific manners are shown as an illustrative description, and can be flexibly selected according to the needs, and the present disclosure is not limited to the above-mentioned manners, which will not be described here.

[0122] The freezing chip of the present disclosure limits the temperature gradient to the part of the heat-conducting layer between the part of the heat-conducting layer close to the temperature control unit and the end part of the heat-conducting layer, thereby limiting the heat capacity of the local temperature control area, so that the freezing speed is higher than 10 5 ℃ / s. For the cell sample, the rapid freezing of the sample does not destroy the cell sample, and is conducive to better study of the cell biological behavior.

[0123] Those skilled in the art can understand that, according to the design needs, the above-mentioned chip substrate, sample placement layer, heat-conducting layer, first isolation layer and second isolation layer in the sample placement layer can be discontinuous, and can be holed, slotted, etc. to adjust the thermal conductivity or facilitate light observation.

[0124] As another embodiment, the sample placement layer 12 comprises: at least one sample layer, a heating layer, a fourth isolation layer, a heat-conducting layer and a fifth isolation layer arranged separately; wherein the surface of the sample layer is divided into at least one local temperature control area; and the temperature control unit is arranged on the heating layer.

[0125] As another embodiment, the sample placement layer 12 comprises: at least one sample layer, a heating layer, a fourth isolation layer, a heat-conducting layer and a fifth isolation layer arranged separately; wherein the surface of the sample layer is divided into at least one local temperature control area; and the temperature control unit is arranged on the heating layer. Figures 2a-2eThe sample placement layer 12 is different from the sample placement layer shown in the figure in that it is in a non-integrated structure as a whole. In use, the sample layer, the heating layer, the fourth isolation layer, the heat conduction layer, and the fifth isolation layer are stacked in sequence and fixed by an external clamp. The sample layer is independently arranged with the other layers. The heating layer, the fourth isolation layer, the heat conduction layer, and the fifth isolation layer can be independently arranged with each other or combined by two or three layers or the like by using a chip micro-nano processing technology. When combined, the combination should be in the stacking order of the sample placement layer in use. Since the sample layer can be independently arranged, the number of sample layers can be flexibly arranged as needed, and the sample layer can be replaced in time when it is damaged. Compared with the sample placement layer in an integrated structure, the sample placement layer in a split structure will generate new thermal resistance between layers, which will usually affect the freezing speed of the freezing chip. The freezing chip provided in the embodiment of the present disclosure can reduce the influence of the interlayer thermal resistance on the freezing speed when the heat is conducted to the low-temperature cold source A in the transverse direction. Through tests, the freezing speed can also be realized in the order of 10 5 ℃ / s.

[0126] It should be noted that other technical details of the sample layer, the heating layer, the fourth isolation layer, the heat conduction layer, and the fifth isolation layer can refer to the embodiments of the sample placement layer shown in the figure, such as the sample layer corresponding to the isolation layer for placing the sample; the heating layer corresponding to the isolation layer provided with the temperature control unit; the fourth isolation layer corresponding to the first isolation layer, used for isolating the temperature control unit and the heat conduction layer; and the fifth isolation layer corresponding to the third isolation layer, used for isolating the chip substrate and the heat conduction layer, which will not be described here. Figures 2e-2d

[0127] In addition, the freezing chip provided in the present disclosure can also be improved in the following aspects:

[0128] a. The thickness of the part of the heat conduction layer is greater than the thickness of the part of the heat conduction layer between the part close to the temperature control unit and the end part of the heat conduction layer.

[0129] b. The part of the heat conduction layer between the part close to the temperature control unit and the end part of the heat conduction layer is arranged in a patterned structure, such as the part connecting the part close to the temperature control unit and the end part of the heat conduction layer in a radial channel manner.

[0130] Specifically, Figure 3 A schematic diagram of the temperature gradient in the sample placement layer according to the embodiment of the present disclosure is shown. As Figure 3 ​As shown, the temperature of the low-temperature cold source A is -170℃, and the temperature of point w1 at the bottom of the chip substrate 11 is approximately the same as the temperature of the low-temperature cold source A, for example, -160℃. The temperature of point w2 at the top of the sample placement layer 12 is, for example, -120℃. Point w3, which is located on the same plane as w2 and is close to the temperature control unit 13, has a temperature of, for example, 30℃ when the temperature control unit heats the sample. Therefore, the temperature gradient is mainly concentrated between points w3 and w2. The above temperature values ​​are for illustrative purposes only and do not constitute a limitation of this disclosure.

[0131] The inventors discovered that the freezing rate is limited by the heat capacity of the local temperature-controlled region. Since the final freezing temperature of the sample is fixed, minimizing the relatively high-temperature region before freezing—for example, by making the local temperature-controlled region sufficiently small and placing the temperature control unit as close to the sample as possible—can limit the heat capacity of the local temperature-controlled region and improve the freezing rate. On the other hand, using a structure with relatively low thermal conductivity in a location near the temperature control unit outside of it can concentrate the temperature gradient as close to the unit as possible. For example, concentrating the temperature gradient between points w3 and w4, rather than between w3 and w2, can also improve the freezing rate. Combining these two improvements is beneficial for increasing the freezing rate.

[0132] Improving the cryo-chip using methods a and / or b described above can further enhance its freezing speed. Testing has shown that a freezing speed of 10 [units unspecified] can be achieved. 5 On the order of ℃ / s.

[0133] Figure 4 A schematic diagram of a cryogenic chip for placing samples according to an embodiment of the present disclosure is shown. Figure 4 As shown, with Figure 1a The difference is that the local temperature control area is provided with at least one closed sample receiving cavity a and / or an open sample receiving cavity b for accommodating samples. Of course, it is also possible to... Figures 1b-1c The cryogenic chip shown is provided with a closed sample receiving cavity a and / or an open sample receiving cavity b, but this disclosure does not limit this. For other technical details of the cryogenic chip in the embodiments of this disclosure, please refer to... Figures 1a-1c The embodiments shown are not described in detail here.

[0134] According to embodiments of this disclosure, the temperature control unit 13 further includes an auxiliary temperature control unit disposed on the wall of the closed sample receiving cavity a and / or the open sample receiving cavity b, for reducing the temperature difference between multiple samples placed in the same local temperature control area. In this embodiment, the auxiliary temperature control unit and the temperature control unit may use the same components or equivalent components.

[0135] Figure 5 A schematic diagram of the sample stage assembly according to an embodiment of the present disclosure is shown. Figure 5As shown, the sample stage assembly 20 comprises: the frozen chip 10, a sample heat sink 21 and a controller 22. The sample heat sink 21 is used to accommodate the frozen chip 10. The controller 22 is electrically connected with the temperature control unit 13, and is used to adjust the temperature of the temperature control unit 13. It should be noted that the sample heat sink 21 can be designed as a light-transmitting structure to adapt to the observation of the sample by the microscope.

[0136] In the present disclosure, the sample heat sink 21 in the sample stage assembly 20 can be regarded as a low-temperature cold source A. It can be understood that the sample heat sink 21 can also be omitted, and the frozen chip 10 is directly placed on the heat sink base 32 described below, at this time, the heat sink base 32 can be regarded as a low-temperature cold source A, and the present disclosure does not limit this.

[0137] In the present disclosure, the sample stage assembly 20 further comprises a control circuit board (not shown in the figure), which can be embedded in the sample heat sink 21 or arranged around the area of the sample heat sink 21 in direct contact with the frozen chip 10, so as not to affect the efficient heat transfer between the two, and the present disclosure does not limit the position of the control circuit board. The controller 22 is electrically connected with the temperature control unit 13 through the control circuit board, and then adjusts the temperature of the temperature control unit 13.

[0138] Figure 6 A structural schematic diagram of a freezing system according to an embodiment of the present disclosure is shown. As shown in the figure, Figure 6 The freezing system 30 comprises: a sample stage assembly 20, a low-temperature cold source 31 and a heat sink base 32. The low-temperature cold source 31 can be liquid nitrogen, which is used to cool and keep the heat sink base 32 close to the temperature of liquid nitrogen. The heat sink base 32 is used to fix the sample stage assembly 20, and serves as a cold source to freeze the sample stage assembly 20.

[0139] According to an embodiment of the present disclosure, when freezing the sample, the heat sink base 32 is in direct contact with the sample heat sink 21, so that the temperature of the sample heat sink 21 is close to or equal to the temperature of liquid nitrogen, and other parts of the sample stage assembly 20 except the local temperature control area N are also frozen at the same time. The controller 22 adjusts the electrical parameters of the temperature control unit 13, and the sample is directly cooled by the other parts of the chip and the sample heat sink 21 whose surrounding temperature is close to or equal to the temperature of liquid nitrogen.

[0140] According to an embodiment of the present disclosure, the freezing system 30 further comprises a frozen medium sealing cover plate 33, which is used to seal the low-temperature cold source, and in some cases can also support the heat sink base 32 to be immersed in the low-temperature cold source.

[0141] According to an embodiment of the present disclosure, the freezing system 30 further comprises a sample cover plate 34, which has an area 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, which is arranged to ensure that no water vapor enters the low-temperature environment where the freezing chip is located, preventing water vapor from condensing to form liquid droplets adhering to the sample, thereby avoiding the formation of ice crystals in the liquid droplets in the low-temperature environment affecting 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 is generally capable of sealing the low-temperature environment where the freezing chip is located to prevent water vapor from entering, and on this basis, the length of the sample cover plate 34 can be appropriately increased, which is not limited in the present disclosure.

[0142] In the present disclosure, the sample cover plate 34 can also be provided with an observation area or a detection area, so that the sample can be microscopically observed through the observation area and / or the properties of the sample can be characterized by using a detection device at the position of the detection area under the premise of preventing water vapor from entering the low-temperature environment. In some cases, a dry atmosphere can be provided for the low-temperature environment to solve the defect that water vapor condensation affects sample observation or characterization, and at this time, the sample cover plate 34 can be omitted.

[0143] The present disclosure also provides a sample testing system, which comprises the freezing system 30 and a microscopic observation device and / or a detection device used in conjunction with the freezing system 30.

[0144] According to an embodiment of the present disclosure, the microscopic observation device is at least one of an upright optical microscope, an inverted optical microscope, and an electron microscope. The detection device is at least one of a photodetector, X-ray, a Raman spectrometer, an infrared spectrometer, and other monitoring instruments.

[0145] Figure 7 A flowchart of a method for freezing a sample according to an embodiment of the present disclosure is shown. As shown in the figure, the method uses the freezing system 30 to freeze the sample, which comprises the following steps S110-S140. Figure 7

[0146] In step S110, the temperature of the local temperature control area is adjusted to a first temperature;

[0147] In the present disclosure, first, the control circuit board is connected with 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 measuring the resistance value in real time, such as 30℃), and is kept constant at this temperature (adjusted by resistance feedback), because the distance between the temperature control unit and the sample is extremely small and the thermal resistance is extremely low, so it can be approximately considered that the temperature of the sample is also at the set temperature (such as 30℃), and the typical resistance value at this time is in the range of Rheater=50-100 ohm.

[0148] ​In step S120, the sample is placed in the local temperature control region;

[0149] In step S130, the electrical parameter of the temperature control unit is adjusted 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;

[0150] In the present disclosure, when the sample stage assembly is placed on the heat sink base after freezing (about -190°C), the temperature of the frozen chip begins to decrease, at this time, the controller automatically increases the current Iheater for resistance heating, and the average temperature of the sample in the local temperature control region N is maintained at the first temperature (such as 30°C), and the typical current value is in the range of Iheater=50-100 mA, and the typical power of Rheater (Rheater*Iheater2) is about 0.3 W;

[0151] In step S140, the electrical parameter is 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 in the lowest temperature range that can be provided by the low-temperature cold source.

[0152] In the present disclosure, when freezing is required, the controller sends a signal to suddenly reduce the current Iheater to 0.1-1.0 mA, and the temperature of the sample in the local temperature control region N rapidly decreases to the temperature of the heat sink base 32, and Rheater also rapidly decreases to about 1 / 7 of Rheater at room temperature, and during the entire cooling process, the control circuit maintains a small constant current (0.1-1.0 mA). 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 the second temperature (such as -190°C), and the change of Rheater is continuously monitored to serve as a reference for the temperature of the sample.

[0153] In the present disclosure, the second temperature is determined according to the temperature of the low-temperature cold source A, and the sample temperature can be adjusted to the required temperature as long as it is not lower than the temperature. Specifically, when the low-temperature cold source A can provide a low temperature of -190°C, the sample temperature can be adjusted to the required temperature, such as -140°C.

[0154] It should be noted that steps S110 and S120 are steps performed before the sample stage assembly is placed in the heat sink base, and in step S110, the temperature of the local temperature control region can also be room temperature, and at this time, the controller does not need to start heating the temperature control unit. In addition, the execution order of steps S110 and S120 can be interchanged, and the present disclosure does not limit this.

[0155] The basic principle of the operation of the temperature control unit is described as follows:

[0156] Figure 8 The basic principle of the working of the temperature control unit according to the embodiment of the present disclosure is shown in a schematic diagram. Referring to FIG. 1, the temperature control unit is connected in a 4-terminal measurement mode, i.e., Force_H(I+), Sense_H(V+), Sense_L(V-), Force_L(I-). A heating current Iheater is applied from I+ to I-, and the current can reach a maximum of 50-200 mA. At the same time, the voltage difference Vheater is measured between V+ and V-, and the port current at the two ends is very small (such as a virtual ground), which has no effect on the current passing through the temperature control unit. The resistance value Rheater of the temperature control unit is measured in real time by Vheater / Iheater, and the average temperature of the temperature control unit is evaluated by the resistance value Rheater. Figure 8

[0157] It should be noted that in the embodiment of the present disclosure, the function of local selected freezing can be realized by controlling the corresponding temperature control unit of different local temperature control areas. The temperature control unit and the local temperature control area can be in a one-to-one correspondence, and of course, one temperature control unit can be used to adjust the temperature of multiple local temperature control areas according to needs. Those skilled in the art can freely combine, and the above-mentioned methods can be used to realize the function of rapid freezing of samples. The present disclosure does not limit this.

[0158] According to the embodiment of the present disclosure, the average temperature of the sample is adjusted by adjusting the electrical parameter. The electrical parameter can be a current, a resistance, or a power parameter, and the present disclosure does not limit this.

[0159] In the present disclosure, the temperature control unit can be used to measure the temperature of the sample in real time while heating the sample, or a temperature measurement unit can be additionally arranged on the freezing chip. The temperature control unit is used to heat the sample, and the temperature measurement unit is used to measure the temperature of the sample in real time. The present disclosure does not limit this.

[0160] In the present disclosure, the resistance-time curve can be drawn, and then the sample cooling speed can be evaluated according to the resistance-time curve. Specifically, the Iheater current is kept unchanged, Rheater is calculated by measuring Vheater, and the Rheater-time curve during the cooling process is continuously monitored. The curve can be used as a reference for evaluating the sample freezing speed.

[0161] According to the embodiment of the present disclosure, the first temperature is changed to the second temperature within a predetermined time period.

[0162] In the present disclosure, the predetermined time period for reducing the first temperature to the second temperature is controlled to be within 10 ms, for example, 1-2 ms. Specifically, within 1 ms, the temperature is reduced from room temperature to below -140℃, and then further reduced to below -180℃ within the subsequent 1-2 ms. ​

[0163] According to an embodiment of the present disclosure, the time delay can be the delay time of the control system from sending the electrical signal for reducing the first temperature to the refrigeration chip receiving the electrical signal and starting to freeze the sample. It can be understood that when testing a biological sample, it is necessary to determine the time point of freezing the biological sample to observe the sample at the time point or to 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 frozen sample is close to the state of the sample at the time of freezing operation, thereby better performing sample testing.

[0164] According to an embodiment 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.

[0165] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, for example, the temperature of an aqueous solution under normal pressure, which is in the range of 0-40°C for a conventional cell sample, preferably 20-30°C; for special heat-resistant cells or bacteria, the temperature can be increased; and under the condition of high pressure, the temperature range can also change to ensure that the culture solution is in a liquid state and the biological sample is alive.

[0166] According to an embodiment of the present disclosure, the second temperature is the temperature that makes the same sample directly change from a liquid state to an amorphous solid state under the same environment and continuously maintain the amorphous solid state, for example, for water or a general aqueous solution, the temperature should be lower than -140°C, and when the pressure is high or low, the temperature range can change to ensure that the culture solution is frozen to a temperature at which the amorphous state is stable, so as not to destroy the structure of the sample.

[0167] Figure 9 A flowchart of a method for heating a sample according to an embodiment of the present disclosure is shown. As shown in Figure 9 The method uses the refrigeration system 30 to heat the sample, which includes the following steps S210-S220.

[0168] In step S210, the electrical parameters are detected and adjusted to make the average temperature of the local temperature control region to the second temperature.

[0169] In the present disclosure, first, the temperature control unit is connected with the controller under the condition of low temperature (liquid nitrogen temperature); second, the control circuit is started, the I_Heater set value is 0.1-1.0 mA (only for measuring the resistance value to evaluate the temperature, and the heating can be ignored), and the temperature of the temperature control unit is close to the temperature of the heat sink.

[0170] In step S220, the electrical parameter of the temperature control unit is adjusted to a second predetermined range, and then the electrical parameter is detected and adjusted to maintain the average temperature of the sample at the first temperature; or the sample is heated by an external heat source, and the average temperature of the sample is determined by the temperature measuring unit to be at the first temperature; wherein the first temperature is greater than the second temperature.

[0171] In the present disclosure, when the temperature of the temperature control unit is close to the temperature of the heat sink, the IHeater is suddenly increased to make the Rheater heat to the Rheater value corresponding to the set temperature (such as 30°C) at the fastest speed. In this process, since the initial Rheater is the resistance value at the temperature of liquid nitrogen, which is only about 1 / 7 of the room temperature, the initial heating current will reach the order of magnitude of 200-300 mA to reach the power of 0.3 W, so as to achieve the purpose of rapid heating. At the same time, during the heating process, the Iheater needs to be quickly adjusted (decreased) to a reasonable range, so as to maintain the Rheater at the set value (such as the Rheater corresponding to 30°C) all the time. Then the heating element is maintained at the set temperature (such as 30°C), and the sample can be removed or continue to be frozen according to the need.

[0172] In the present disclosure, the external heat source can be used to define the heating area on the freezing chip as a local temperature control area by focusing, heat the sample, and then realize the control of the heating power and the temperature by cooperating with the feedback system on the freezing chip, such as setting a temperature measuring unit on the freezing chip to monitor the sample temperature in real time, and then controlling the heating power of the external heat source. The external heat source can be microwave, laser, etc.

[0173] It should be noted that after the sample is frozen by using the freezing chip, step S210 can be omitted, and step S220 of heating the sample is directly executed.

[0174] The method for heating the sample provided by the embodiments of the present disclosure uses the freezing system 30 to heat the sample, and the specific technical details are referred to the embodiments shown in Figure 6 The embodiments of the present disclosure are not described here.

[0175] According to the embodiments of the present disclosure, the second temperature is increased to the first temperature within a predetermined time period.

[0176] According to the embodiments of the present disclosure, the predetermined time period is within 10 ms, for example, 1-2 ms.

[0177] According to an embodiment of the present disclosure, the first temperature is a liquid temperature of the sample, for example, a water solution under normal pressure, and for a normal cell sample, the temperature is in a range of 0-40℃, preferably 20-30℃; for special heat-resistant cells or bacteria, the temperature can be increased; under high pressure, the temperature range can also change to ensure that the culture solution is in a liquid state and the biological sample is normally alive.

[0178] According to an embodiment of the present disclosure, the second temperature is a temperature at which the same sample is directly converted from a liquid state to an amorphous solid state under the same environment and continuously maintained in the amorphous solid state, for example, for water or a general water solution, the temperature should be lower than -140℃, and when the pressure is high or low, the temperature range can change to ensure that the culture solution is frozen to a temperature at which the amorphous state is stable, so as not to destroy the sample structure.

[0179] Figure 10 A flowchart of a method for operating a sample according to an embodiment of the present disclosure is shown. As shown in the flowchart, the method uses a sample testing system to operate a sample, including the following steps S310-S370. Figure 10

[0180] 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 the temperature gradient of the sample in the sample placement layer and the low-temperature cold source is maintained.

[0181] In step S320, the electrical parameters are detected and adjusted to a first predetermined range to adjust the average temperature of the sample to the 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 in the lowest temperature range that can be provided by the low-temperature cold source.

[0182] In step S330, the electrical parameters of the temperature control unit are adjusted to a second predetermined range to heat the sample or use an external heat source to heat the sample to the first temperature, 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.

[0183] In step S340, after the sample is operated, the sample is replaced.

[0184] ​It should be noted that step S340 can be performed after the sample is heated to the first temperature in step S320, that is, after the sample is operated once at the second temperature, the sample is heated to the first temperature, and then the sample is frozen as needed, the sample is operated twice, the sample is heated to the first temperature, and then the operation is ended. The disclosure does not limit the number of cycles of freezing, heating, and re-freezing. It can be understood that after the operation is ended in step S320, a new sample can also be replaced at the first temperature, and then the new sample is frozen and operated repeatedly. The disclosure does not limit this.

[0185] The method for operating a sample provided by the embodiments of the disclosure will be described in detail with reference to the embodiments shown in Figure 7 、 Figure 9 The method for operating a sample provided by the embodiments of the disclosure will be described in detail with reference to the embodiments shown in

[0186] According to the embodiments of the disclosure, the operation of the sample can be the detection signal of the sample under the monitoring instrument such as a micro-observation sample, a photoelectric detector, an X-ray, a Raman spectrometer, and an infrared spectrometer. The disclosure does not limit this.

[0187] According to the embodiments of the disclosure, after the step of 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 of the sample and the low-temperature heat source in the sample placement layer in step S310, the method further comprises:

[0188] Operating the sample at the first temperature and determining the starting moment of adjusting the electrical parameters to the first predetermined range, at the starting moment, detecting and adjusting the electrical parameters to the first predetermined range to maintain the average temperature of the sample at the second temperature.

[0189] According to the embodiments of the disclosure, the first temperature is changed to the second temperature within a first predetermined time period.

[0190] According to the embodiments of the disclosure, the electrical parameters of the temperature control unit are adjusted by an electronic device. For example, by using keithley 2612B to adjust the electrical parameters of the temperature control unit, the time delay can be controlled to be less than 2 ms.

[0191] According to the embodiments of the 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.

[0192] According to the embodiments of the disclosure, the second temperature is changed to the first temperature within a second predetermined time period.

[0193] According to the embodiments of the disclosure, the second predetermined time period is less than 10 ms, for example, 1-2 ms.

[0194] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, for example, the temperature of an aqueous solution under normal pressure, which is in the range of 0-40℃ for a conventional cell sample, preferably 20-30℃, and can be increased for special heat-resistant cells or bacteria, and the temperature range can also change under high pressure conditions to ensure that the culture solution is in a liquid state and the biological sample is alive.

[0195] According to an embodiment of the present disclosure, the second temperature is the temperature at which the same sample is directly converted from a liquid state to an amorphous solid state under the same environment and continuously maintained in the amorphous solid state, for example, the temperature of water or an aqueous solution should be lower than -140℃, and the temperature range can change under high or low pressure conditions to ensure that the culture solution is frozen to a temperature at which the amorphous state is stable, so as not to destroy the sample structure.

[0196] The sample testing system according to an embodiment of the present disclosure is used to operate the sample, and by adjusting the parameters of the temperature control unit, the operation process of freezing the sample and operating the sample, or the cycle of the above process of freezing the sample, operating the sample, heating and reviving the sample, freezing the sample, operating the sample, and heating and reviving the sample, or the operation process of operating the sample before freezing, freezing the sample, and operating the sample, or the cycle of the above process of operating the sample before freezing, freezing the sample, operating the sample, heating and reviving the sample, operating the sample before freezing, freezing the sample, operating the sample, and heating and reviving the sample can be realized, and the above process can also be repeated after replacing the sample after freezing the sample and operating the sample. The technical solution limits the heat capacity of the local temperature control area by designing the thermal resistance and heat exchange efficiency of the interfaces between the local temperature control area, the chip substrate, and the low-temperature cold source, and obtains a freezing and heating speed higher than 10 5 ℃ / s, which ensures that the sample is not destroyed (or the damage is reduced) in the repeated freezing and heating process, which is a major improvement for biological sample freezing, in-situ observation, and heating and thawing operations, and has great significance and wide application prospects.

[0197] The following specifically describes the way in which the sample testing system according to an embodiment of the present disclosure is used to observe the sample under a microscope.

[0198] Method one: placing the sample in the local temperature control area, maintaining the temperature to the first temperature, freezing to the second temperature, and observing under a microscope, which is suitable for protein samples and high-resolution microscopic observation after sample freezing and preparation;

[0199] Method two: placing the sample in the local temperature control area, maintaining the temperature to the first temperature, real-time microscopic observation, starting freezing at a specific time node, maintaining the temperature to the second temperature, and high-resolution microscopic observation, which is suitable for cell samples and can first observe the sample activity in real time, freeze the sample at a specific time point of interest, for example, cell division or cell phagocytosis, and then perform high-resolution microscopic observation.

[0200] It should be noted that the microscope used for real-time microscopic observation before freezing and after freezing can be different, so as to realize observation with different resolutions. For example, the sample is observed in real time by using a conventional upright optical microscope, and the cell is observed in high resolution structure after freezing by using an electron microscope.

[0201] The method for microscopic observation of a sample provided by the embodiments of the present disclosure can freeze the cell sample from 20-30℃ to about -170℃ in less than 2 ms, and the freezing speed is higher than 10 5 ℃ / s, so that the cell sample remains substantially unchanged in shape after freezing, neither broken nor obviously deformed.

[0202] The above description is merely preferred embodiments of the present disclosure and a description of the principles of the technology used. Those skilled in the art should understand that the scope of the application involved in the present disclosure is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by any combinations of the above technical features or equivalent features without departing from the inventive concept. For example, the above features are replaced with each other to form a technical solution with similar functions disclosed in the present disclosure (but not limited to).

Claims

1. A cryogenic chip, characterized in that, The cryogenic chip is in contact with a low-temperature cold source to freeze samples, including: A sample placement layer, the surface of which is divided into at least one local temperature control area, the local temperature control area being used to place the sample; Several temperature control units are used to independently adjust the temperature of each of the local temperature control zones or to jointly adjust the temperature of several of the local temperature control zones; The chip substrate supports the top or bottom surface of the sample placement layer to form a first contact surface; the projection of the first contact surface and the local temperature control area on the same plane does not overlap or partially overlaps.

2. The cryogenic chip according to claim 1, characterized in that, The chip substrate is supported in the peripheral region outside the central region of the sample placement layer; the central region is divided into at least one local temperature control region; or The chip substrate is supported on the central region of the sample placement layer; the peripheral region outside the central region is divided into at least one local temperature control region; or The chip substrate is supported at intervals within the local temperature control area.

3. The cryogenic chip according to claim 1, characterized in that, When the chip substrate supports the top surface of the sample placement layer to form the first contact surface, the chip substrate also has a second contact surface for contacting the low-temperature cold source; wherein the first contact surface and the second contact surface are located on the same side of the chip substrate.

4. The cryogenic chip according to claim 1, characterized in that, The temperature control unit and the sample placement layer are an integrated structure.

5. The cryogenic chip according to claim 1, characterized in that, The temperature control unit is fabricated on the sample placement layer using chip micro-nano fabrication technology, and the temperature control unit is used to divide the local temperature control area.

6. The cryogenic chip according to claim 5, characterized in that, The sample placement layer is a thermally conductive layer; the temperature control unit is disposed on the thermally conductive layer to divide the local temperature control area on the thermally conductive layer; or The sample placement layer includes: a thermally conductive layer and a first isolation layer fabricated on the thermally conductive layer using chip micro / nano fabrication technology; wherein, the temperature control unit is disposed on the first isolation layer to divide the local temperature control area on the first isolation layer; or The sample placement layer includes: a thermally conductive layer, a first isolation layer fabricated on the thermally conductive layer using chip micro / nano fabrication technology, and a second isolation layer fabricated on the first isolation layer using chip micro / nano fabrication technology; wherein, the temperature control unit is disposed on the first isolation layer to divide the local temperature control area on the second isolation layer; or The sample placement layer includes: a third isolation layer, a thermally conductive layer fabricated on the third isolation layer using chip micro / nano fabrication technology, a first isolation layer fabricated on the thermally conductive layer using chip micro / nano fabrication technology, and a second isolation layer fabricated on the first isolation layer using chip micro / nano fabrication technology; wherein, the temperature control unit is disposed on the first isolation layer to divide the local temperature control area on the second isolation layer; or The sample placement layer includes: a third isolation layer, a first isolation layer fabricated on the third isolation layer using chip micro-nano fabrication technology, a thermally conductive layer fabricated on the first isolation layer using chip micro-nano fabrication technology, and a second isolation layer fabricated on the thermally conductive layer using chip micro-nano fabrication technology; wherein, the temperature control unit is disposed on the third isolation layer to divide the local temperature control area on the second isolation layer.

7. The cryogenic chip according to claim 1, characterized in that, The sample placement layer includes: at least one sample layer, a heating layer, a fourth isolation layer, a thermally conductive layer, and a fifth isolation layer, which are separately configured; The sample layer surface is divided into at least one local temperature control area; the temperature control unit is disposed on the heating layer.

8. The cryogenic chip according to claim 6 or 7, characterized in that, The thickness of the portion of the heat-conducting layer near the temperature control unit and the end portion of the heat-conducting layer is greater than the thickness of the portion of the heat-conducting layer between them; and / or The portion of the heat-conducting layer near the temperature control unit and the portion at the end of the heat-conducting layer are arranged in a patterned structure.

9. The cryogenic chip according to any one of claims 6-7, characterized in that, The local temperature control area is provided with at least one closed sample receiving cavity and / or an open sample receiving cavity for accommodating samples.

10. The cryogenic chip according to claim 9, characterized in that, The temperature control unit also includes an auxiliary temperature control unit disposed on the wall of the closed sample receiving cavity and / or the open sample receiving cavity.

11. The cryogenic chip according to any one of claims 1-7 and 10, characterized in that, The sample placement layer is equipped with optical pathways to accommodate microscopes, photodetectors, X-ray spectrometers, Raman spectrometers, and infrared spectrometers.

12. The cryogenic chip according to claim 11, wherein the cryogenic chip is made of a light-transmitting material or has a perforated channel as the light path channel.

13. The cryogenic chip according to any one of claims 1-7, 10, and 12, characterized in that, The cryogenic chip is fabricated using chip micro-nano fabrication technology.

14. The cryogenic chip according to claim 13, characterized in that, The thickness of the cryogenic chip is controlled between 0.1 and 2 mm.

15. A sample stage assembly comprising a cryogenic chip as described in any one of claims 1-14, characterized in that, include: A controller electrically connected to the temperature control unit is used to adjust the temperature of the temperature control unit.

16. The sample stage assembly according to claim 15, characterized in that, Also includes: A sample heat sink is used to house the frozen chip.

17. A refrigeration system comprising the sample stage assembly as described in claim 15 or 16, characterized in that, include: Low-temperature cold source; The heat sink base of the sample stage assembly is fixed in contact with the low-temperature cold source.

18. The refrigeration system according to claim 17, characterized in that, Also includes: A freezing medium sealing cover, which is used to seal the low-temperature cold source.

19. The refrigeration system according to claim 17, characterized in that, Also includes: The sample cover plate has an area sufficient to seal the opening of the heat sink base.

20. A sample testing system comprising a freezing system as described in any one of claims 17-19, characterized in that, include; Microscopic observation and / or detection devices used in conjunction with the freezing system.

21. The sample testing system according to claim 20, characterized in that, The microscopic observation device is at least one of an upright optical microscope, an inverted optical microscope, and an electron microscope; The detection device is at least one of a photodetector, an X-ray, a Raman spectrometer, and an infrared spectrometer.

22. A method for freezing samples using the freezing system as described in any one of claims 17-19, characterized in that, include: Adjust 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. 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 desired temperature value is determined within the lowest temperature range that the low-temperature cold source can provide.

23. The method according to claim 22, characterized in that, Before adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample stable at a first temperature and to maintain the temperature gradient between the sample and the low-temperature cold source within the sample placement layer, the method further includes: Adjust the temperature of the local temperature control area to the first temperature; Place the sample within the local temperature-controlled area.

24. The method according to claim 22, characterized in that, Change the first temperature to the second temperature within a predetermined time period.

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

26. The method according to claim 22, characterized in that, The electrical parameters of the temperature control unit are adjusted via electronic devices.

27. The method according to claim 22, 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 a liquid state to an amorphous solid state under the same environment and maintained as an 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 a freezing system as described in any one of claims 17-19, characterized in that, include: Detect and adjust electrical parameters to bring the average temperature of the local temperature control area to a second temperature; 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 by a temperature measuring unit at a first temperature; wherein the first temperature is greater than the second temperature.

30. The method according to claim 29, characterized in that, Change the second temperature to the first temperature within a predetermined time period.

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

32. The method according to claim 29, 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 a liquid state to an amorphous solid state under the same environment and maintained as an 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 as described in claim 20, characterized in that, include: Adjust 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 within the sample placement layer; The electrical parameters are 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 desired temperature value is determined within the lowest temperature range that the low-temperature cold source can provide.

35. The method according to claim 34, characterized in that, Also includes: 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 a first temperature using an external heat source. Then, the electrical parameters are repeatedly detected and adjusted to the first predetermined range to maintain the average temperature of the sample at a second temperature. The sample is then operated at the second temperature.

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

37. The method according to claim 36, characterized in that, Also includes: After handling the sample, replace the sample.

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

39. The method according to claim 34, characterized in that, The electrical parameters of the temperature control unit are adjusted via electronic devices.

40. The method according to claim 34, characterized in that, The second temperature is changed to the first temperature within a second predetermined time period.

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

42. The method according to any one of claims 34-35 and 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 a liquid state to an amorphous solid state under the same environment and maintained as an 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, characterized in that, The method described is applicable to microscopic observation of samples.

Images