CN112747995A

CN112747995A - Freezing chip, freezing system, sample testing system and sample testing method

Info

Publication number: CN112747995A
Application number: CN202011583914.7A
Authority: CN
Inventors: 赵蒙
Original assignee: Zhuhai Sade Technology Co ltd; Bioisland Laboratory
Current assignee: Zhuhai Sade Technology Co ltd; Bioisland Laboratory
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-05-04
Also published as: CN116685838A; CN214668100U; WO2022142061A1

Abstract

The embodiment of the disclosure discloses a freezing chip, a freezing system, a sample testing system and a sample testing method, wherein the freezing chip comprises a chip substrate; the surface of the sample placing layer is divided into at least one local temperature control area, and the local temperature control area is used for placing a sample; the temperature control units are used for adjusting the temperature of the local temperature control area; wherein the chip substrate is provided with a supporting surface for supporting a partial area of the top surface or the bottom surface of the sample placing layer to form a first contact surface; the projection of the area of the first contact surface and the local temperature control area on the same plane is not completely overlapped. The technology can select a specific time to freeze and unfreeze in the process of in-situ observation and characterization of a sample, and the interface thermal resistance is higher than 10⁵The freezing and heating speed of the temperature is controlled to ensure that the sample is frozen at the temperature of DEG C/sIs not damaged. The method is a great improvement on the related operations of biological sample freezing, thawing, in-situ microscopic observation and the like, and has great significance and wide application prospect.

Description

Freezing chip, freezing system, sample testing system and sample testing method

Technical Field

The disclosure relates to the technical field of biomedicine, in particular to a freezing chip, a freezing system, a sample testing system and a sample testing method.

Background

The rapid freezing and heating technology of biological samples has many important applications in the biomedical field, such as cell freezing storage and revival, protein freezing and fixing characterization, and the like.

The current biological freezing techniques mainly include plug-in freezing, spray freezing and high-pressure freezing. Plug-in freezing (plug freeze) is the most common sample preparation method in the industry at present. The plug-in freezing is usually accomplished by fixing a sample stage (micro-grid) carrying a biological sample at the front end of a sample rod, and rapidly inserting the sample into a cryogenic liquid, such as liquid ethane or liquid nitrogen, by mechanical control. In jet freezing (freezing), a biological sample is generally frozen by transporting a sample stage carrying the biological sample to a specific position in a freezing chamber through a sample rod and then jetting the sample at high speed using high-pressure liquid nitrogen vapor. High pressure freeze (high pressure freeze) is similar to the principle of plug-in freezing, a low-temperature liquid is adopted to freeze a sample, high pressure of about 2000 atmospheric pressure is applied to a sample cavity during freezing, the freezing temperature of water is reduced, and volume expansion generated in the ice crystallization process is inhibited, so that the damage of ice crystallization on the structure of a biological sample is avoided, and a frozen biological sample with high quality is prepared.

However, the plug-in freezing has the following drawbacks: because the sample needs to be inserted into the cryogenic liquid in its entirety, it is not possible to selectively freeze a specific region of the sample during freezing, nor to perform real-time microscopic observation in situ during freezing. The jet freezing is based on the plug-in freezing, and liquid nitrogen steam is used for replacing low-temperature liquid, so that the heat transfer efficiency is improved. The principle of high-pressure freezing is similar to that of the two freezing modes, and due to the fact that ice crystallization is inhibited through high pressure, the freezing effect is good, and the quality of a sample is high. However, spray freezing and high-pressure freezing also have the disadvantage that they are not amenable to real-time microscopic observation and localized selective freezing. These deficiencies limit further intensive research into freezing biological samples.

There is also provided in the prior art an apparatus for rapid freezing of a sample, the apparatus comprising: the sample container is placed on the base, and the sample is quickly frozen by controlling the switch of the heating supporting device. The device has the advantage that the heating and supporting device is arranged on the side surface of the sample container, so that the freezing speed of the frozen sample is not ideal. In addition, in the aspect of recovering and freezing a biological sample by heating, the conventional method has a low heating speed at present, and usually auxiliary media such as DMSO (dimethyl sulfoxide) and the like need to be added into the sample to ensure that the biological sample is not damaged in the heating process, so that the biological sample has an influence on the activity of the biological sample, cannot express the real performance of the biological sample such as cells and the like in a normal environment.

Disclosure of Invention

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

In a first aspect, embodiments of the present disclosure provide a frozen chip.

Specifically, the freezing chip is in contact with a low-temperature cold source for freezing a sample, and comprises: the surface of the sample placing layer is divided into at least one local temperature control area, and the local temperature control area is used for placing a sample; a plurality of temperature control units for adjusting the temperature of the local temperature control area; the chip substrate supports the top surface or the bottom surface of the sample placement layer to form a first contact surface; the first contact surface and the local temperature control area are not overlapped or partially overlapped in projection on the same plane.

Optionally, the chip substrate is supported in a peripheral area outside a central area of the sample placement layer, and the central area is divided into at least one local temperature control area; or the chip substrate is supported in the central area of the sample placement layer, and the peripheral area outside the central area is divided into at least one local temperature control area; or the chip substrate is supported at spaced locations of the local temperature controlled zone.

Optionally, when the top surface of the chip substrate supporting the sample placement layer forms the first contact surface, the chip substrate further has a second contact surface thereon for contacting with 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.

Optionally, the temperature control unit and the sample placement layer are of an integrated structure.

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

Optionally, the sample placement layer is a heat conducting layer; the temperature control unit is arranged on the heat conduction layer to divide the local temperature control area on the heat conduction layer; or

The sample placement layer includes: the heat conduction layer and a first isolation layer are manufactured on the heat conduction layer by adopting a chip micro-nano processing technology; the temperature control unit is arranged on the first isolation layer to divide the local temperature control area on the first isolation layer; or

The sample placement layer includes: the heat conduction layer, a first isolation layer manufactured on the heat conduction layer by adopting a chip micro-nano processing technology and a second isolation layer manufactured on the first isolation layer by adopting the chip micro-nano processing technology; the temperature control unit is arranged on the first isolation layer to divide the local temperature control area on the second isolation layer; or

The sample placement layer includes: the heat conduction layer is manufactured on the third isolation layer by adopting a chip micro-nano processing technology, the first isolation layer is manufactured on the heat conduction layer by adopting the chip micro-nano processing technology, and the second isolation layer is manufactured on the first isolation layer by adopting the chip micro-nano processing technology; the temperature control unit is arranged on the first isolation layer to divide the local temperature control area on the second isolation layer; or

The sample placement layer includes: the heat conduction layer is manufactured on the first isolation layer by adopting a chip micro-nano processing technology, and the second isolation layer is manufactured on the heat conduction layer by adopting the chip micro-nano processing technology; wherein the temperature control unit is disposed on the third isolation layer to divide the local temperature control region on the second isolation layer.

Optionally, the sample placement layer comprises: the device comprises at least one sample layer, a heating layer, a fourth isolating layer, a heat conducting layer and a fifth isolating layer which are arranged in a split mode; wherein the surface of the sample layer is divided into at least one local temperature control area; the temperature control unit is arranged on the heating layer.

Optionally, the thickness of the part of the heat conduction layer close to the temperature control unit and the end part of the heat conduction layer is larger than that of the part of the heat conduction layer between the heat conduction layer and the heat conduction layer; and/or the heat conducting layer part between the part of the heat conducting layer close to the temperature control unit and the end part of the heat conducting layer is arranged in a patterned structure.

Optionally, the local temperature controlled zone is provided with at least one closed sample receiving chamber for receiving a sample and/or an open sample receiving chamber.

Optionally, the temperature control unit further comprises an auxiliary temperature control unit disposed on a wall of the closed sample-receiving chamber and/or the open sample-receiving chamber.

Optionally, the sample placement layer is provided with an optical channel to adapt to a microscope, a photodetector, X-ray, raman spectrometer, infrared spectrometer.

Optionally, the freezing chip is made of a light-transmissive material or has a perforated channel as the light passage channel.

Optionally, the frozen chip is manufactured by a chip micro-nano processing technology.

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

In a second aspect, embodiments of the present disclosure provide a sample stage assembly including a frozen chip according to any one of the first aspects. Specifically, the sample stage assembly includes: and the controller is electrically connected with the temperature control unit and is used for adjusting the temperature of the temperature control unit.

Optionally, the sample stage assembly further comprises: a sample heat sink for receiving the freezing chip.

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

Optionally, the refrigeration system further comprises:

and the freezing medium sealing cover plate is used for sealing the low-temperature cold source.

Optionally, the refrigeration system further comprises:

a sample cover plate having an area capable of sealing at least the opening of the heat sink base.

In a fourth aspect, embodiments of the present disclosure provide a sample testing system comprising the refrigeration system of the third aspect. Specifically, the sample testing system comprises;

a microscopic observation device and/or a detection device used with the freezing system.

Optionally, the microscopic observation device is at least one of an upright optical microscope, an inverted optical microscope and an electronic microscope; the detection device is at least one of a photoelectric detector, an X ray, a Raman spectrometer and an infrared spectrometer.

In a fifth aspect, embodiments of the present disclosure provide a method of freezing a sample using the freezing system of the third aspect. Specifically, the method comprises the following steps: adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample to be stabilized at a first temperature and maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer; and detecting and adjusting the electrical parameter to a first preset range so as to adjust the average temperature of the sample to be at a second temperature, wherein the second temperature is lower than the first temperature, and a required temperature value is determined in a lowest temperature range which can be provided by the low-temperature cold source.

Optionally, before adjusting the electrical parameter of the temperature control unit to maintain the average temperature of the sample stable at the first temperature and maintain the temperature gradient between the sample and the low-temperature heat sink in the sample placement layer, the method further includes: adjusting the temperature of the local temperature control area to a first temperature; and placing a sample in the local temperature control area.

Optionally, the first temperature is changed to the second temperature within a predetermined period of time.

Optionally, the electrical parameters of the temperature control unit are adjusted by electronic means.

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

Optionally, the first temperature is from 0 ℃ to 40 ℃ and the second temperature is less than-140 ℃.

In a sixth aspect, embodiments of the present disclosure provide a method of heating a sample using the freezing system of the third aspect. Specifically, the method comprises the following steps: adjusting the electrical parameter of the temperature control unit to a second preset range, and then detecting and adjusting the electrical parameter to maintain the average temperature of the sample at a first temperature; or an external heat source is used for heating the sample, and the average temperature of the sample is determined to be the first temperature through a temperature measuring unit; wherein the first temperature is greater than the second temperature.

Optionally, the method further comprises:

and detecting and adjusting the electrical parameter to enable the average temperature of the local temperature control area to reach a second temperature.

Optionally, the second temperature is increased to the first temperature for a predetermined period of time.

Optionally, the predetermined time period is within 10 ms.

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 a liquid state to an amorphous state in the same environment and the amorphous state is continuously maintained.

In a seventh aspect, embodiments of the present disclosure provide a method of operating a sample using the sample testing system of the fourth aspect.

Specifically, the method comprises the following steps: adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at a first temperature and maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer; detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, and thereafter operating the sample at the second temperature, wherein the second temperature is lower than the first temperature, determining a desired temperature value within a minimum temperature range that the low temperature heat sink is capable of providing.

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

Optionally, the method further comprises: after the steps of adjusting the electrical parameter 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 in the sample placement layer, operating the sample at the first temperature and determining a starting moment when the electrical parameter is adjusted to a first preset range, and detecting and adjusting the electrical parameter to the first preset range at the starting moment to maintain the average temperature of the sample at a second temperature.

Optionally, the method further comprises: after manipulating the sample, the sample is replaced.

Optionally, the first temperature is changed to the second temperature within a first predetermined period of time.

Optionally, the second temperature is changed to the third temperature within a second predetermined period of time.

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

Alternatively, the method is suitable for microscopic observation of a sample.

The technical scheme provided by the embodiment of the disclosure can have the following beneficial effects:

(1) the freezing chip of the embodiment of the disclosure, through setting up at least one local temperature control area, utilize the temperature control unit to adjust the temperature in local temperature control area, can selectively freeze the sample, to the sample that does not need freezing, control the temperature control unit release heat in order to maintain the temperature gradient of sample and low temperature cold source, to the sample that needs freezing, adjust the electrical parameter of temperature control unit to make sample heat conduct to low temperature cold source, thereby realized the frozen effect in local election district.

(2) The freezing chip of this disclosure embodiment, the layer integration setting is placed with the sample to control by temperature change unit, when freezing chip and low temperature cold source contact, places the temperature gradient that the in situ formed sample and low temperature cold source at the sample, and through the electricity parameter of adjustment control by temperature change unit, the sample heat can be fast along the conduction of temperature gradient direction to realize the quick freezing of sample, can provide low temperature system appearance for other testing arrangement, for example microscope, X ray apparatus etc..

(3) The freezing chip of the embodiment of the disclosure, through designing the structure of the heat conduction layer, limits the temperature gradient at the heat conduction layer part between the part of the heat conduction layer close to the temperature control unit and the end part of the heat conduction layer, reduces the heat capacity of the frozen part while ensuring the heat transfer speed, and ensures that the freezing speed is higher than 10⁵For cell samples, the rapid freezing of the sample does not destroy the cell sample, facilitating better study of cell biological behavior.

(4) According to the freezing chip disclosed by the embodiment of the disclosure, the sample placing layer is provided with the optical path channel, so that the sample can be adapted to a testing device to carry out in-situ characterization on the sample, such as a microscope, an X-ray device and the like, the sample can be tested in situ in real time while the sample is frozen, and the sample testing efficiency is improved.

(5) The sample testing system of the embodiment of the present disclosure is used for a method for operating a sample, by adjusting parameters of a temperature control unit, a cycle of the above procedures of freezing a sample-operating a sample, or freezing a sample-operating a sample-heating and reviving a sample-freezing a sample-operating a sample-heating and reviving a sample can be realized, or a cycle of the above procedures of operating a sample before freezing, freezing a sample-operating a sample-heating and reviving a sample-freezing a sample-operating a sample-heating and reviving a sample can be realized, or the above procedures can be repeated after the sample is frozen and the sample is replaced. The technical scheme limits the heat capacity of the local temperature control area through the design of the heat resistance and the heat exchange efficiency of each interface among the local temperature control area, the chip substrate and the low-temperature cold source, and obtains the heat capacity higher than 10⁵The freezing and heating speed of the temperature/s ensures that the structure and the function of the sample are not damaged in the repeated freezing and heating process, which is a great improvement on operations such as biological sample freezing, in-situ observation, heating and thawing and the like, and has great significance and wide application prospect.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

Drawings

Other features, objects, and advantages of the disclosure will become apparent from the following detailed description of non-limiting embodiments, which proceeds with reference to the accompanying drawings. In the drawings:

FIG. 1a shows a front view of a cryochip according to an embodiment of the disclosure;

FIG. 1b shows a cross-sectional view in the direction DD' in FIG. 1 a;

FIG. 1c shows a cross-sectional view of a cryochip according to another embodiment of the present disclosure;

FIG. 1d shows a cross-sectional view of a cryochip according to another embodiment of the present disclosure;

2 a-2 e show schematic structural views of a sample placement layer according to embodiments of the present disclosure;

FIG. 3 shows a schematic diagram of a temperature gradient within a sample placement layer according to an embodiment of the present disclosure;

FIG. 4 shows a schematic structural diagram of a freezing chip on which a sample is placed according to an embodiment of the present disclosure;

FIG. 5 shows a schematic structural view of a sample stage assembly according to an embodiment of the disclosure;

FIG. 6 shows a schematic structural diagram of a refrigeration system according to an embodiment of the present disclosure;

FIG. 7 shows a schematic flow diagram of a method of freezing a sample according to an embodiment of the present disclosure;

FIG. 8 illustrates a basic schematic diagram of the operation of a temperature control unit according to an embodiment of the present disclosure;

FIG. 9 shows a schematic flow diagram of a method of heating a sample according to an embodiment of the present disclosure;

FIG. 10 shows a schematic flow diagram of a method of microscopically observing a sample according to an embodiment of the present disclosure;

FIG. 11 shows a schematic of a chip and cell samples before and after on-chip freezing.

Detailed Description

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

In the present disclosure, it is to be understood that terms such as "including" or "having," etc., are intended to indicate the presence of the disclosed features, numbers, steps, behaviors, components, parts, or combinations thereof, and are not intended to preclude the possibility that one or more other features, numbers, steps, behaviors, components, parts, or combinations thereof may be present or added.

It should be noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

In the prior art, the plug-in refrigeration has the following defects: because the sample needs to be inserted into the cryogenic liquid in its entirety, it is not possible to selectively freeze a specific region of the sample during freezing, nor to perform real-time microscopic observation in situ during freezing. The jet freezing is based on the plug-in freezing, and liquid nitrogen steam is used for replacing low-temperature liquid, so that the heat transfer efficiency is improved. The principle of high-pressure freezing is similar to that of the two freezing modes, and due to the fact that ice crystallization is inhibited through high pressure, the freezing effect is good, and the quality of a sample is high. However, spray freezing and high-pressure freezing also have the defect that real-time microscopic observation and local selective freezing cannot be realized. These deficiencies limit further intensive research into freezing biological samples. Meanwhile, no mature technology capable of high-speed heating to recover the frozen sample is available at present.

The present disclosure is made to solve, at least in part, the problems in the prior art that the inventors have discovered.

The freezing chip provided by the present disclosure is different from the three ways of plug-in freezing, spray freezing and high-pressure freezing in the principle of freezing the sample. The difference is that the sample placed on the freezing chip is not in direct contact with a freezing medium (such as liquid nitrogen), but the sample is kept at a higher temperature by adopting an external resistance heating mode while the freezing medium cools a sample stage (the chip is usually placed on the sample stage, and the sample stage is immersed in the freezing medium). After the resistance heating is closed, the heat of the sample is quickly transferred to the low-temperature sample stage, so that the sample is quickly frozen.

Fig. 1a shows a front view of a cryochip according to an embodiment of the disclosure. The low temperature cold source a shown in fig. 1b-1d is not a part of the freezing chip 10, and the low temperature cold source a in the present disclosure 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 low temperature cold source a 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, so that the sample heat sink also has a temperature of the low temperature cold source (for example, liquid nitrogen), and may be regarded as the low temperature cold source a. The above is a schematic illustration, and the disclosure does not limit the low temperature cold source a.

As shown in fig. 1a to 1d, the freezing chip 10 includes: a chip substrate 11, a sample placement layer 12 and a number of temperature control units 13. The chip substrate 11 is in contact with the top or bottom surface of the supporting 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 controlled area N for placing a sample. The temperature control unit 13 generates heat locally, typically by joule heating (heat is generated by an electric current through a resistor), to adjust the temperature of the local temperature controlled area N. The area P of the first contact surface 14 and the projection of the local temperature control area N on the same plane do not overlap or partially overlap.

According to the embodiment of the present disclosure, the top surface of the sample placement layer 12 is used for placing a sample, the chip substrate 11 generally supports the bottom surface of the sample placement layer 12 to form a first contact surface 14 (as shown in fig. 1 b), the chip substrate 11 can also support the top surface of the sample placement layer 12 to form a first contact surface (as shown in fig. 1 c), fig. 1d also shows the top surface of the chip substrate 11 supporting the sample placement layer 12, and unlike fig. 1c, the chip substrate 11 further has a second contact surface for contacting with the low temperature heat sink a when the top surface of the chip substrate 11 supporting the sample placement layer 12 forms the first contact surface; the first contact surface and the second contact surface are positioned on the same side of the chip substrate.

The description shown in fig. 1b to 1d is a schematic description, and can be flexibly selected according to the needs, and the disclosure is not limited to the above setting manner, and is not repeated herein.

According to embodiments of the present disclosure, the same plane may be the plane in which the sample placement layer 12 is located. As shown in fig. 1b, the region P of the first contact surface 14 does not overlap with the local temperature control region N, and the sample heat in the local temperature control region N is conducted from the local temperature control region N to the region P along the direction indicated by the arrow, and then conducted to the low temperature heat sink a along the chip substrate 11. In fig. 1c, the region P partially overlaps with the local temperature control region N, and the sample heat in the local temperature control region N1 is conducted from the local temperature control region N1 to the region P1 along the direction indicated by the arrow, and then conducted to the low-temperature heat sink a along the chip substrate 11, unlike in fig. 1b, the sample heat in the local temperature control region N2 is conducted from the local temperature control region N2 to the low-temperature heat sink a along the direction indicated by the arrow, and then conducted through the temperature control unit 13 along the longitudinal direction of the local temperature control region N2.

It should be noted that the wires of the temperature control unit 13 may be connected to the controller outside the freezing chip 10 through the sample placing layer 12, and the amount of heat generated by the wires is negligible when the temperature of the sample in the local temperature control region N is adjusted.

In the freezing chip shown in fig. 1b-1d, the sample heat can be conducted to the low-temperature heat source a along the transverse conduction and longitudinal conduction directions, so as to freeze the sample. Furthermore, the central region of the chip substrate 11 is hollow, and can be adapted to a testing device for in situ characterization of a sample, such as a microscope, an X-ray device, and the like, without limitation to the present disclosure.

When the freezing chip provided by the disclosure is used, before a sample is frozen, the freezing chip is placed on the low-temperature cold source A, the temperature control unit 13 maintains the sample at a first temperature, such as 20 ℃ to 30 ℃, and at this time, the temperature gradient between the sample and the low-temperature cold source A is formed in the sample placing layer. After the freezing starts, the electrical parameters of the temperature control unit 13 are adjusted, the heat of the sample in the local temperature control area N is conducted along the direction of the temperature gradient, so as to realize the rapid freezing of the sample, and then the electrical parameters are detected to adjust the temperature of the sample to a required second temperature, for example, when the low-temperature cold source a can provide a low temperature of-190 ℃, the temperature of the sample can be adjusted to-140 ℃.

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 by the present disclosure.

The freezing chip of the embodiment of the disclosure, through setting up at least one local temperature control area, utilize the temperature control unit to adjust the temperature in local temperature control area, can selectively freeze the sample, to the sample that does not need to freeze, control the temperature control unit release heat in order to maintain the temperature gradient of sample and low temperature cold source, for the sample that needs to freeze, adjust the electrical parameter of temperature control unit to make sample heat conduct to low temperature cold source, thereby realized the frozen effect in local election district.

According to the embodiment of the present disclosure, the chip substrate 11 is supported in the peripheral region outside the central region of the sample placement layer 12; the central region of the sample placement layer 12 is divided into at least one local temperature controlled zone. For example, the chip substrate 11 is a surrounding structure adapted to the periphery outside the central region of the sample placement layer 12, and surrounds and supports the top surface or the bottom surface of the sample placement layer 12; or the chip substrate 11 is an independent supporting block supported on one side or both sides of the central region, etc.; wherein the central area is divided into at least one local temperature controlled area N. The upper or lower portion of the central region may be adapted to allow in situ characterization of the sample by a testing device, such as a microscope, X-ray device, or the like.

In another embodiment, the chip substrate 11 is supported on the central region of the sample-placing layer 12; the peripheral area outside the central area is divided into at least one local temperature control area N. For example, the frozen chip is T-shaped, the sample-placing layer 12 is horizontally disposed, and the region supported by the chip base 11 is not used to divide the local temperature-controlled region N, but is divided into several local temperature-controlled regions N around the support region.

In some cases, the chip substrate 11 may also be supported at spaced locations of the local temperature control region N. For example, the chip substrate 11 is at least two independent supporting blocks for supporting the sample placement layer 12, and the local temperature control region N may be divided in the region between the supporting blocks and the peripheral region outside the supporting blocks.

According to an embodiment of the present disclosure, the frozen chip 10 is manufactured by a chip micro-nano processing technology, for example, a thin film deposition technology, a dry or wet etching technology, a photolithography technology, and the like in the field of chips, which are not described herein again.

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

According to the embodiment of the disclosure, the sample placement layer 12 is provided with the light passage, so that the sample can be adapted to a testing device to perform in-situ characterization on the sample, such as a microscope, an X-ray device, and the like, thereby realizing in-situ real-time sample testing while freezing the sample, and improving the sample testing efficiency. Specifically, the freezing chip is made of a light-transmitting material or is provided with a perforated channel as the light passage channel so as to be suitable for monitoring instruments such as an upright optical microscope, an inverted optical microscope, an electron microscope, a photoelectric detector, an X-ray, a Raman spectrometer and an infrared spectrometer.

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

According to the embodiment of the disclosure, the temperature control unit 13 is disposed in the sample placement layer 12 by a chip micro-nano processing technology, and the local temperature control area N of the local temperature control area 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 perform heating control and stop heating, so as to independently adjust the temperature of the samples placed in different local temperature control regions N, and in some cases, the temperatures of the samples in several local temperature control regions N can also be jointly adjusted, which is not limited by the present disclosure.

According to the 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 metal (aluminum, copper, platinum, etc.), metal compound (titanium nitride, indium tin oxide, etc.) or semiconductor (silicon, silicon carbide, etc.).

Fig. 2 a-2 e show schematic structural views of a sample placement layer according to embodiments of the present disclosure. As shown in fig. 2 a-2 e, 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 heat conducting layer 121 is used for transversely conducting sample heat to the low-temperature heat source a, the first isolation layer 122 is used for isolating the heat conducting layer 121 and the temperature control unit 13, the second isolation layer 123 is used for isolating the temperature control unit 13 from an external contact environment, and plays roles of insulating and protecting the temperature control unit 13, and the third isolation layer 124 is used for isolating the chip substrate 11 from the heat conducting layer 121. The first isolation layer 122, the second isolation layer 123, and the third isolation layer 124 may be omitted as appropriate.

The material of the heat conduction layer 121 may be metal (e.g., aluminum, copper, platinum, etc.), heat conduction ceramic (e.g., aluminum oxide, aluminum nitride, etc.), or other heat conduction materials (e.g., silicon carbide, silicon nitride, etc.). The thickness of the thermally conductive layer 121 is typically 0.1-5 um.

According to the embodiment of the present disclosure, the temperature control unit 13 and the sample placement layer 12 are an integrated structure.

As shown in fig. 2a, the sample placement layer 12 includes only a heat conductive layer 121; the temperature control unit 13 is disposed on the heat conductive layer 121 to divide the local temperature control area N on the heat conductive layer 121. In this embodiment, the sample-placing layer 12 is composed of only the heat-conducting layer, and the power consumption for maintaining the sample temperature is large, but the freezing speed of the freezing chip can be up to 10 with a high freezing speed⁵-10⁶℃/s。

As shown in fig. 2b, the sample placement layer 12 includes: the heat conduction layer 121 and the first isolation layer 122 manufactured on the heat conduction layer 121 by adopting a chip micro-nano processing technology; wherein the temperature control unit 13 is disposed on the first isolation layer 122 to divide the local temperature control region on the first isolation layer 122. In this embodiment, the freezing speed of the freezing chip is lower than that of FIG. 2a, and still can reach 10⁵-10⁶℃/s。

As shown in fig. 2c, the sample placement layer 12 includes: the heat conduction layer 121, the first isolation layer 122 manufactured on the heat conduction layer 121 by adopting a chip micromachining process and the second isolation layer 123 manufactured on the first isolation layer 122 by adopting the chip micromachining process; 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, the second isolation layer 123 is disposed on the first isolation layer 122, so as to prevent the temperature control unit 13 from being exposed to the external environment, and to prolong the service life of the frozen chip, and after testing, the freezing speed of the frozen chip can still reach 10⁵-10⁶℃/s。

As shown in fig. 2d, the sample placement layer 12 comprises: the heat conduction layer is characterized by comprising a third isolation layer 124, a heat conduction layer 121 manufactured on the third isolation layer 124 by adopting a chip micro-nano processing technology, a first isolation layer 122 manufactured on the heat conduction layer 121 by adopting the chip micro-nano processing technology, and a second isolation layer 123 manufactured on the first isolation layer 122 by adopting the chip micro-nano processing 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, the third isolation layer 123 is disposed under the heat conducting layer 121, and considering that the heat conducting layer 121 is usually made of metal material, based on the convenience of the processing technology, the third isolation layer 123 may be disposed between the heat conducting layer 121 and the chip substrate 11, so as to meet the technological requirements, and through the test, the freezing speed of the frozen chip may still reach 10⁵℃/s。

As shown in fig. 2e, the sample placement layer 12 includes: the heat conduction layer comprises a third isolation layer 124, a first isolation layer 122 manufactured on the third isolation layer 124 by adopting a chip micro-nano processing technology, a heat conduction layer 121 manufactured on the first isolation layer 122 by adopting the chip micro-nano processing technology, and a second isolation layer 123 manufactured on the heat conduction layer 121 by adopting the chip micro-nano processing technology; wherein the temperature control unit 13 is disposed on the third isolation layer 124 to divide the local temperature control region on the second isolation layer 123. In this embodiment, different from the embodiment in fig. 2d, the temperature control unit 13 is located below the heat conducting layer 121 and is closer to the chip substrate 11 and the low temperature heat sink a, so that the power consumption is larger, and after testing, the freezing speed of the frozen chip can still reach 10⁵℃ /s。

In fig. 2a to 2e, the heat conductive layer 121 is preferably made of a high thermal conductive material, such as a metal material, to increase the freezing speed.

The specific manner shown above is used as an illustrative description, and can be flexibly selected according to needs, and the disclosure is not limited to the above manner, and is not repeated herein.

Cooling of embodiments of the present disclosureThe chip is frozen, and the structure of the heat conduction layer is designed to limit the temperature gradient at the heat conduction layer part between the part of the heat conduction layer close to the temperature control unit and the end part of the heat conduction layer, so that the heat capacity of a local temperature control area is limited, and the freezing speed is higher than 10⁵For cell samples, the rapid freezing of the sample does not destroy the cell sample, facilitating better study of cell biological behavior.

It can be understood by those skilled in the art that, depending on the design requirements, the chip substrate, the sample placement layer, the heat conduction layer in the sample placement layer, the first isolation layer, and the second isolation layer may be discontinuous, and holes, slots, etc. may be formed therein to adjust the heat conductivity or facilitate the observation of transmitted light.

As another embodiment, the sample placement layer 12 includes: the device comprises at least one sample layer, a heating layer, a fourth isolating layer, a heat conducting layer and a fifth isolating layer which are arranged in a split mode; wherein the surface of the sample layer is divided into at least one local temperature control area; the temperature control unit is arranged on the heating layer.

Different from the sample-placing layer shown in fig. 2a to 2e, the sample-placing layer 12 is a non-integrated structure, and when in use, the sample layer, the heating layer, the fourth isolation layer, the heat-conducting layer, and the fifth isolation layer are sequentially stacked and fixed by an external clamp. The sample layer and other layers are independently arranged, the heating layer, the fourth isolating layer, the heat conducting layer and the fifth isolating layer can be independently arranged, two layers or three layers can be combined by adopting a chip micro-nano processing technology, and the like, and the sample layer and the fifth isolating layer are combined according to the stacking sequence when the sample layer is used. Because the sample layers can be independently arranged, the number of the sample layers can be flexibly arranged according to the requirement, and when a certain sample layer is damaged, the sample layer can be replaced in time. Compared with the sample placing layer with an integrated structure, the sample placing layer with a split structure can generate new thermal resistance between layers, and the freezing speed of the freezing chip can be influenced. When the heat of the freezing chip provided by the embodiment of the disclosure is conducted to the low-temperature cold source A along the transverse direction, the influence of interlayer thermal resistance on the freezing speed can be reduced, and through tests, the freezing speed can also be 10⁵Number of DEG C/sMagnitude.

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 be referred to the embodiments of the sample placement layer shown in fig. 2e-2d, for example, the sample layer corresponds to the isolation layer for placing the sample; the heating layer corresponds to the isolation layer provided with the temperature control unit; the fourth isolating layer corresponds to the first isolating layer and is used for isolating the temperature control unit from the heat conducting layer; the fifth isolation layer corresponds to the third isolation layer and is used for isolating the chip substrate from the heat conduction layer, which is not described herein again.

In addition, the freezing chip provided by the present disclosure can be improved from the following aspects:

a, adjusting the thickness of the heat conduction layer part, wherein the thickness of the part close to the temperature control unit and the thickness of the end part of the heat conduction layer are larger than the thickness of the heat conduction layer part between the temperature control unit and the heat conduction layer;

b 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 is arranged in a patterned structure, for example, the part connects the part close to the temperature control unit and the end part of the heat conducting layer in a radial channel mode.

In particular, fig. 3 shows an illustrative diagram of a temperature gradient within a sample placement layer according to an embodiment of the disclosure. As shown in FIG. 3, the temperature of the low temperature heat sink A is-170 deg.C, and the temperature at point w1 on the bottom of the chip substrate 11 is approximately 160 deg.C, for example. The temperature at point w2 on top of the sample placement layer 12 is for example-120 ℃. The temperature at point w3, which is located on the same plane as the point of location w2 and is close to the temperature control unit 13, is, for example, 30 ℃ when the temperature control unit heats the sample, and the temperature gradient is mainly concentrated between the point w3 and the point w 2. The above temperature values are illustrative and do not constitute a limitation of the present disclosure.

The inventors have found that the freezing rate is limited by the heat capacity of the local temperature controlled zone. Since the final freezing temperature of the sample is determined, the relatively high temperature region before freezing is minimized, for example, the range of the local temperature control region is small enough, and the temperature control unit is as close to the sample as possible, so as to limit the heat capacity of the local temperature control region, and increase the freezing speed. On the other hand, a structure with relatively low thermal conductivity is adopted at a position which is outside the temperature control unit and is close to the temperature control unit, so that the temperature gradient is concentrated in an area close to the temperature control unit as much as possible, for example, the temperature gradient is concentrated between a point w3 and a point w4 instead of between a point w3 and a point w2, and the freezing speed is improved. The improvement of the two aspects is combined, so that the freezing speed is favorably improved.

The freezing chip is improved by the mode a and/or the mode b, the freezing speed of the freezing chip can be further provided, and the freezing speed can be 10 times after being tested⁵In the order of DEG C/s.

Fig. 4 shows a schematic structural diagram of a freezing chip on which a sample is placed according to an embodiment of the present disclosure. As shown in fig. 4, unlike fig. 1a, the local temperature controlled area is provided with at least one closed sample-receiving chamber a and/or an open sample-receiving chamber b for receiving a sample. Of course, a closed sample-receiving chamber a and/or an open sample-receiving chamber b may be provided on the basis of the cryochip shown in FIGS. 1b-1c, which is not limited by the present disclosure. Other technical contents of the freezing chip of the embodiment of the disclosure are shown in the embodiment portions shown in fig. 1a to fig. 1c, and are not repeated herein.

According to the embodiment of the present disclosure, the temperature control unit 12 further includes an auxiliary temperature control unit disposed on the wall of the closed sample-accommodating cavity a and/or the open sample-accommodating cavity b, for reducing the temperature difference between the multiple samples placed in the same local temperature control region. In the present embodiment, the auxiliary temperature control unit and the temperature control unit may employ the same components or equivalent components.

Fig. 5 shows a schematic structural diagram of a sample stage assembly according to an embodiment of the disclosure. As shown in fig. 5, the sample stage assembly 20 includes: a freezing chip 10, a sample heat sink 21 and a controller 22. Wherein the sample heat sink 21 is used for accommodating the freezing chip 10. The controller 22 is electrically connected to the temperature control unit 13, and is configured to adjust the temperature of the temperature control unit 13. It should be noted that the sample heat sink 21 can be designed to be a light-transmitting structure to accommodate a microscope for observing the sample.

In the present disclosure, the sample heat sink 21 in the sample stage assembly 20 may be regarded as a low temperature heat sink a. It is understood that the sample heat sink 21 may be omitted and the freezing chip 10 may be directly placed on the heat sink base 32 described below, and in this case, the heat sink base 32 may be regarded as the low temperature heat sink a, which is not limited by the present disclosure.

In the present disclosure, the sample stage assembly 20 further includes a control circuit board (not shown in the figure), and the control circuit board may be embedded in the sample heat sink 21 or disposed around an area where the sample heat sink 21 and the freezing chip 10 are in direct contact, so as not to affect efficient heat transfer of the two, and the present disclosure does not limit the position of the control circuit board. The controller 22 is electrically connected to the temperature control unit 13 through the control circuit board, so as to adjust the temperature of the temperature control unit 13.

Fig. 6 shows a schematic structural diagram of a refrigeration system according to an embodiment of the present disclosure. As shown in fig. 6, the freezing system 30 includes: sample stage assembly 20, low temperature cold source 31 and heat sink base 32. The low temperature cold source 31 may be liquid nitrogen and is used to cool and maintain the heat sink base 32 near the temperature of the liquid nitrogen. The heat sink base 32 is used for fixing the sample stage assembly 20 and is used as a cold source for freezing the sample stage assembly 20.

According to the embodiment of the present disclosure, when the sample is frozen, 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 the same as the temperature of the 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 sample heat sink 21 and other parts of the chip where the ambient temperature is close to or equal to the temperature of the liquid nitrogen.

According to an embodiment of the present disclosure, the refrigeration system 30 further comprises: a freezing medium sealing cover plate 33, wherein the freezing medium sealing cover plate 33 is used for sealing the low-temperature cold source, and in some cases, the heat sink base 32 can be supported to be immersed in the low-temperature cold source.

According to an embodiment of the present disclosure, the refrigeration system 30 further comprises: a sample cover plate 34 having an area at least capable of sealing the opening of the heat sink base 32. The length of the sample cover plate 34 shown in the figure extends to two ends of the freezing medium sealing cover plate 33 respectively, so that the freezing chip is prevented from entering water vapor in the low-temperature environment, the water vapor is prevented from being condensed to form liquid drops to be attached to the sample, and the liquid drops are prevented from forming ice crystals in the low-temperature environment to influence microscopic observation or property characterization of the sample. It will be appreciated that the sample cover 34 is of sufficient area to cover the sample heat sink, and typically seals the low temperature environment in which the frozen chip is located to prevent ingress of moisture, and that the length of the sample cover 34 may be increased appropriately, without limitation.

In the present disclosure, the sample cover 34 may further be provided with an observation region or a detection region, so as to microscopically observe the sample through the observation region and/or characterize the properties of the sample at the detection region by using a detection device under the premise of preventing moisture from entering in the low-temperature environment. In some cases, a dry atmosphere may be provided to the cryogenic environment to address defects in the condensation of water vapor that affect the observation or characterization of the sample, and in such cases, the sample cover 34 may be omitted.

The present disclosure also provides a sample testing system comprising a freezing system 30 and a microscopic observation device and/or a detection device used in conjunction with the freezing system 30.

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 monitoring instruments such as a photoelectric detector, an X-ray, a Raman spectrometer and an infrared spectrometer.

Fig. 7 shows a schematic flow diagram of a method of freezing a sample according to an embodiment of the present disclosure. As shown in FIG. 7, the method utilizes a freezing system 30 to freeze a sample, including the following steps S110-S140.

In step S110, adjusting the temperature of the local temperature control area to a first temperature;

in the present disclosure, first, the control circuit board is connected to the controller at room temperature; next, the controller is activated to heat the temperature control unit to a set temperature slightly above room temperature (the temperature of the temperature control unit is determined by measuring the resistance value in real time, e.g., 30 ℃), and is maintained at that temperature (adjusted by resistance feedback) since the temperature is maintained at that temperatureThe distance between the temperature control unit and the sample is extremely small, the thermal resistance is extremely low, therefore, the sample temperature can be approximately considered to be also at the set temperature (such as 30 ℃), and the typical resistance value range is in R_heater＝50-100ohm。

In step S120, a sample is placed in the local temperature controlled area;

in step S130, 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 between the sample and the low temperature cold source in the sample placement layer;

in the disclosed mode, the sample stage assembly is placed on a heat sink base (about-190 ℃) after freezing, the temperature of the freezing chip begins to decrease, and at the moment, the controller automatically increases the current I_heaterResistance heating is carried out to maintain the average temperature of the sample in the local temperature control area N at a first temperature (such as 30 ℃), and the typical current value ranges from I_heater＝50-100mA，R_heaterTypical power (R)_heater*I_heater ²) About 0.3W;

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 a required temperature value is determined within a lowest temperature range that the low temperature heat sink can provide.

In the disclosed mode, when refrigeration is needed, the controller sends out a signal to enable the current I to be applied_heaterWhen the temperature of the sample in the local temperature control area N is suddenly reduced to 0.1-1.0mA, the temperature of the sample in the local temperature control area N is rapidly reduced to the temperature of the heat sink base 31, R_heaterAlso sharply decreases R to about room temperature_heater1/7, the control circuit maintains a small constant current (0.1-1.0mA) throughout the cool down process. After the cold freezing, the control circuit keeps a small current (0.1-1.0mA), maintains the average temperature of the sample at a second temperature (such as-190 ℃), and continuously monitors R_heaterThe change was used as a reference for the sample temperature.

In this disclosure, the second temperature is determined according to the temperature of the low-temperature cold source a, and is not lower than the temperature. In particular, when the low temperature cold source a can provide a low temperature of-190 ℃, the sample temperature can be adjusted to a desired temperature, for example, can be-140 ℃.

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 area may also be room temperature, and at this time, the controller does not need to be started to heat the temperature control unit. In addition, the execution order of step S110 and step S120 may be interchanged, which is not limited by the present disclosure.

The basic principle of operation of the temperature control unit is explained as follows:

FIG. 8 illustrates a basic schematic diagram of the operation of a temperature control unit according to an embodiment of the present disclosure. Referring to FIG. 8, the temperature control units are connected using 4-terminal measurement, i.e., Force _ H (I +), Sense _ H (V +), Sense _ L (V-), Force _ L (I-). Applying a heating current I through I + to I-_heaterThe maximum current can reach 50-200 mA magnitude. Measuring the voltage difference V simultaneously across V + and V-)_heaterThe port currents at these two ends are small (e.g., virtual ground), and the effect of the current through the temperature control unit is not remembered. Through V_heater/I_heaterReal-time measurement of resistance R of temperature control unit_heaterAnd the average temperature of the temperature control unit is evaluated according to the average temperature.

It should be noted that, in the embodiment of the present disclosure, the function of local area selection freezing may be implemented by controlling the temperature control units corresponding to different local temperature control areas, the temperature control units and the local temperature control areas may be in a one-to-one correspondence relationship, of course, one temperature control unit may also be used to adjust the temperatures of a plurality of local temperature control areas as needed, and those skilled in the art may freely combine the temperature control units and all the temperature control units may also implement the function of rapidly freezing a sample by using the above-mentioned method. The present disclosure is not so limited.

According to an embodiment of the present disclosure, the average temperature of the sample is adjusted by adjusting the electrical parameter. Where the electrical parameter may be a current, resistance, or power parameter, the disclosure is not limited thereto.

In the mode of the present disclosure, the temperature control unit can be used for measuring the temperature of the sample in real time while heating the sample, and the temperature measurement unit can be additionally arranged on the freezing chip, so that the temperature control unit is used for heating the sample, and the temperature measurement unit is used for measuring the temperature of the sample in real time. The present disclosure is not so limited.

In the present disclosure, the resistance may be plotted against time, and then the sample cooling rate may be evaluated based on the resistance versus time. In particular, I can be maintained_heaterBy measuring V with constant current_heaterTo calculate R_heaterContinuously monitoring R in the cooling process_heaterThe time-dependent curve can be used as a reference for evaluating the freezing speed of the sample.

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

In the disclosed manner, the predetermined period of time for lowering the first temperature to the second temperature is controlled to be within 10ms, for example, 1-2 ms. Specifically, the temperature was reduced from room temperature to below-140 ℃ within 1ms, and further reduced to below-180 ℃ within the subsequent 1-2 ms.

According to an embodiment of the present disclosure, the time delay may be a delay time for the control system to begin freezing the sample by sending an electrical signal to lower the first temperature until the freezing chip receives the electrical signal. It will be appreciated that when testing a biological sample, it is necessary to determine the point in time at which the biological sample is frozen, to view the sample at that point in time or to perform other tests. The time delay reflects the delay time of the freezing operation, and the smaller the time delay is, the more accurately the time point of the frozen sample can be controlled, so that the state of the frozen sample is close to the state of the sample during the freezing operation, and the sample test is better.

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

According to an embodiment of the present disclosure, the first temperature is a liquid temperature of the sample, e.g., an aqueous solution at normal pressure, and for a conventional cell sample, the temperature is in the range of 0-40 ℃, preferably 20-30 ℃; for particular heat-resistant cells or bacteria, the temperature may be increased; under non-normal pressure conditions, the temperature range can also be changed to ensure that the culture solution is in a liquid state and the biological sample normally survives.

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

Fig. 9 shows a schematic flow diagram of a method of heating a sample according to an embodiment of the present disclosure. As shown in FIG. 9, the method utilizes the freezing system 30 to heat the sample, including the following steps S210-S220.

In step S210, the electrical parameter is detected and adjusted to make the average temperature of the local temperature controlled area reach the second temperature.

In the present disclosure, first, the temperature control unit is connected to the controller under a low temperature (at a liquid nitrogen temperature); secondly, the control circuit is started, I \ u_HeaterThe set value is 0.1-1.0mA (only for measuring the resistance value to evaluate the temperature, the heating is negligible), and the temperature of the temperature control unit is close to the temperature of the heat sink.

In step S220, adjusting an electrical parameter of the temperature control unit to a second predetermined range, and then detecting and adjusting the electrical parameter to maintain the average temperature of the sample at the first temperature; or an external heat source is used for heating the sample, and the average temperature of the sample is determined to be a first temperature through a temperature measuring unit; wherein the first temperature is greater than the second temperature.

In the mode of the disclosure, when the temperature of the temperature control unit is close to the temperature of the heat sink, the temperature I is suddenly increased_HeaterR at the fastest speed_heaterR corresponding to a set temperature (e.g. 30 ℃ C.) by heating_heaterThe value is obtained. In this process, due to the initial R_heaterThe resistance value at the liquid nitrogen temperature is only about 1/7 times of the room temperature, so that the initial heating current can reach 200-300 mA order and can reach 0.3W equivalent power, thereby achieving the purpose of rapid heating. At the same time, heatingIn the process, the resistance value rapidly rises, I_heaterNeeds to be adjusted (reduced) rapidly to a reasonable range in order to maintain R_heaterAlways at a set value (e.g. R for 30 ℃ C.)_heater). The heating element is then maintained stable at the set temperature (e.g., 30℃.), and the sample can be removed, or continued to freeze, as desired.

In the mode, an external heat source can be utilized to define a heating area in a local temperature control area on a freezing chip through focusing, a sample is heated, and then the heating power and the temperature are controlled through matching with a feedback system on the freezing chip, for example, a temperature measuring unit can be arranged on the freezing chip to monitor the temperature of the sample in real time, and further the heating power of the external heat source is controlled. The external heat source may be microwave, laser, etc.

After the sample is frozen by using the freezing chip, step S210 may be omitted and step S220 may be directly performed to heat the sample.

The method for heating a sample provided in the embodiment of the present disclosure utilizes the freezing system 30 to heat the sample, and specific technical details refer to the embodiment shown in fig. 6, which are not repeated herein.

According to an embodiment of the present disclosure, the second temperature is increased to the first temperature for a predetermined period of time.

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

According to an embodiment of the present disclosure, the first temperature is a liquid temperature of the sample, e.g., an aqueous solution at normal pressure, and for a conventional cell sample, the temperature is in the range of 0-40 ℃, preferably 20-30 ℃; for particular heat-resistant cells or bacteria, the temperature may be increased; under non-normal pressure conditions, the temperature range may also be changed to ensure that the culture solution is in a liquid state and the biological sample is normally alive.

According to the embodiment of the present disclosure, the second temperature is a temperature at which the same sample is directly transformed from a liquid state to an amorphous state in the same environment and the amorphous state is continuously maintained, for example, for water or general aqueous solution, the temperature should be lower than-140 ℃, and the temperature range may be changed at high pressure or low pressure, so as to ensure that the culture solution is frozen to a temperature at which the amorphous state is stable, and thus the structure of the sample is not damaged.

Fig. 10 shows a schematic flow diagram of a method of operating a sample according to an embodiment of the present disclosure. As shown in FIG. 10, the method utilizes a sample testing system to manipulate a sample, including the following steps S310-S370.

In step S310, 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 between the sample and the low temperature cold source in the sample placement layer;

detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, and then operating the sample at the second temperature, wherein the second temperature is lower than the first temperature, and determining a required temperature value within a minimum temperature range that can be provided by the low temperature heat sink in step S320;

in step S330, adjusting an electrical parameter of a temperature control unit to a second predetermined range to heat the sample or heating the sample to a first temperature by using an external heat source, and then repeatedly detecting and adjusting the electrical parameter to the first predetermined range to maintain an average temperature of the sample at a second temperature, and then operating the sample at the second temperature;

in step S340, the sample is replaced after the sample is manipulated.

It should be noted that, step S340 may be performed after heating the sample to the first temperature in step S320, that is, after the sample is operated at the second temperature for the next time, the sample may be repeatedly frozen to the first temperature, and after the sample is operated for the second time, the sample is heated to the first temperature, and then the operation is ended. It is understood that after the operation is ended in step S320, a new sample may be replaced at the first temperature, and then the freezing of the new sample may be repeated, which is not limited by the present disclosure.

Specific technical details of the method for operating a sample provided in the embodiment of the present disclosure refer to the embodiments shown in fig. 7 and fig. 9, which are not repeated herein.

According to the embodiment of the present disclosure, the operation sample may be a detection signal of a microscopic observation sample or a test sample under monitoring instruments such as a photodetector, an X-ray, a raman spectrometer, an infrared spectrometer, and the like, which is not limited by the present disclosure.

According to an embodiment of the present disclosure, 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 between the sample and the low temperature heat source in the sample placement layer in step S310, the method further includes:

operating the sample at a first temperature and determining a start-up time for adjusting the electrical parameter to a first predetermined range, at which time the electrical parameter is detected and adjusted to the first predetermined range to maintain the average temperature of the sample at a second temperature.

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

According to an embodiment of the present disclosure, an electrical parameter of the temperature control unit is adjusted by an electronic device. The time delay can be controlled to within 2ms, for example, by adjusting the electrical parameters of the temperature control unit using keithley 2612B.

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

According to an embodiment of the disclosure, the second predetermined period of time is within 10ms, e.g. 1-2 ms.

The sample testing system of the embodiment of the disclosure is used for a method for operating a sample, and by adjusting the parameters of the temperature control unit, the cycle of the above-mentioned procedures of freezing a sample-operating a sample, or freezing a sample-operating a sample-heating and reviving a sample-freezing a sample-operating a sample-heating and reviving a sample can be realized, or the cycle of the above-mentioned procedures of operating a sample before freezing-freezing a sample-operating a sample, or operating a sample before freezing-freezing a sample-operating a sample-heating and reviving a sample can also be realized by replacing a sample after freezing a sample-operating a sample and then repeating the above-mentioned procedures. The technical scheme limits the heat capacity of the local temperature control area through the design of the heat resistance and the heat exchange efficiency of each interface among the local temperature control area, the chip substrate and the low-temperature cold source, and obtains the heat capacity higher than 10⁵The freezing and heating speed of the temperature/s ensures that the sample is not damaged (or the damage is reduced) in the repeated freezing and heating process, which is a great improvement on operations such as biological sample freezing, in-situ observation, heating and thawing and the like, and has great significance and wide application prospect.

The manner in which the sample testing system provided by embodiments of the present disclosure is used to microscopically observe a sample is described in detail below.

The method comprises the steps of placing a sample in a local temperature control area, keeping the temperature to be the first temperature, freezing to be the second temperature, and observing in a microscopic mode, wherein the method is suitable for protein samples, and high-resolution microscopic observation is carried out after the samples are frozen and prepared;

the second method comprises the following steps: the method is suitable for cell samples, and can be used for firstly observing the movement of the sample in real time, freezing the sample when the cell phagocytoses foreign substances and then carrying out high-resolution microscopic observation.

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

The embodiments of the present disclosure provide a method for microscopic examination of a sample, wherein a cell sample is frozen from 20-30 ℃ to about-170 ℃ in less than 2ms, and the freezing rate is higher than 10⁵And the temperature/s ensures that the shape of the cell sample is basically kept unchanged after the cell sample is frozen, and the cell sample cannot crack or obviously deform.

The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be understood by those skilled in the art that the scope of the invention in the present disclosure is not limited to the specific combination of the above-mentioned features, but also encompasses other embodiments in which any combination of the above-mentioned features or their equivalents is possible without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) technical features disclosed in the present disclosure having similar functions.

Claims

1. A freezing chip, wherein the freezing chip is in contact with a low temperature cold source for freezing a sample, comprising:

the surface of the sample placing layer is divided into at least one local temperature control area, and the local temperature control area is used for placing a sample;

the temperature control units are used for adjusting the temperature of the local temperature control area;

the chip substrate supports the top surface or the bottom surface of the sample placement layer to form a first contact surface; the first contact surface and the local temperature control area are not overlapped or partially overlapped in projection on the same plane.

2. Freezing chip according to claim 1,

the chip substrate is supported in a peripheral area outside a central area of the sample placement layer; the central area is divided into at least one local temperature control area; or

The chip substrate is supported on the central area of the sample placing layer; the peripheral area outside the central area is divided into at least one local temperature control area; or

The chip substrate is supported at spaced locations of the local temperature controlled zone.

3. Freezing chip according to claim 1,

when the top surface of the chip substrate supporting the sample placement layer forms the first contact surface, a second contact surface is further arranged on the chip substrate and is used for contacting with the low-temperature cold source; the first contact surface and the second contact surface are positioned on the same side of the chip substrate.

4. Freezing chip according to claim 1,

the temperature control unit and the sample placing layer are of an integrated structure.

5. A sample stage assembly comprising a frozen chip as claimed in any one of claims 1 to 4, comprising:

and the controller is electrically connected with the temperature control unit and is used for adjusting the temperature of the temperature control unit.

6. A freezing system comprising the sample stage assembly of claim 5, comprising:

a low-temperature cold source;

and the heat sink base is used for fixing the sample stage assembly and is in contact with the low-temperature cold source.

7. A sample testing system comprising the freezing system of claim 6, comprising;

8. A method for freezing a sample using a freezing system according to claim 6, comprising:

adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample to be stabilized at a first temperature and maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer;

detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and determining a required temperature value within a lowest temperature range that the low temperature heat sink can provide.

9. A method for heating a sample with a freezing system according to claim 6, comprising:

adjusting the electrical parameter of the temperature control unit to a second predetermined range, and then detecting and adjusting the electrical parameter to maintain the average temperature of the sample at the first temperature; or an external heat source is used for heating the sample, and the average temperature of the sample is determined to be the first temperature through a temperature measuring unit; wherein the first temperature is greater than the second temperature.

10. A method of manipulating a sample according to the sample testing system of claim 7, comprising:

adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at a first temperature and maintain the temperature gradient between the sample and the low-temperature cold source in the sample placement layer;

detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, and thereafter operating the sample at the second temperature, wherein the second temperature is lower than the first temperature, determining a desired temperature value within a minimum temperature range that the low temperature heat sink is capable of providing.