CN116685838A - Freezing chip, freezing system, sample testing system and method - Google Patents

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

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Publication number
CN116685838A
CN116685838A CN202180086244.4A CN202180086244A CN116685838A CN 116685838 A CN116685838 A CN 116685838A CN 202180086244 A CN202180086244 A CN 202180086244A CN 116685838 A CN116685838 A CN 116685838A
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temperature
sample
layer
temperature control
chip
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赵蒙
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Zhuhai Sade Technology Co ltd
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Zhuhai Sade Technology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G05—CONTROLLING; REGULATING
    • G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
    • G05D23/00—Control of temperature
    • G05D23/19—Control of temperature characterised by the use of electric means
    • G05D23/30—Automatic controllers with an auxiliary heating device affecting the sensing element, e.g. for anticipating change of temperature
    • G05D23/32—Automatic controllers with an auxiliary heating device affecting the sensing element, e.g. for anticipating change of temperature with provision for adjustment of the effect of the auxiliary heating device, e.g. a function of time

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Automation & Control Theory (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

The embodiment of the disclosure discloses a freezing chip, a freezing system, a sample testing system and a 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; the chip substrate is provided with a supporting surface, and a first contact surface is formed by a partial area of the top surface or the bottom surface of the sample placing layer; 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 specific time to freeze and defrost in the process of in-situ observation and characterization of the sample, and obtain the temperature higher than 10 through interface thermal resistance design 5 The freezing and heating speeds at the temperature of/s ensure that the sample is not damaged. The method is a great improvement for related operations such as 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 method
Cross Reference to Related Applications
The present application claims priority from chinese patent application number "CN 202011583914.7" filed on 12/28 in 2020, the entire contents of which are incorporated herein as a whole.
Technical Field
The disclosure relates to the biomedical technology field, in particular to a freezing chip, a freezing system, a sample testing system and a method.
Background
Rapid freezing and heating techniques for biological samples have many important applications in biomedical fields, such as cell frozen storage and rejuvenation, protein frozen fixation characterization, and the like.
The prior biological freezing technology mainly comprises insert freezing, jet freezing and high-pressure freezing. Plug-in freezing (plungge freeze) is the most commonly used sample preparation method in the industry today. Insert freezing typically secures a sample stage (micro-grid) carrying a biological sample to the front end of a sample rod, and the sample is quickly inserted into a cryogenic liquid, such as liquid ethane, or liquid nitrogen, by mechanical control, thereby completing the freezing of the biological sample. Spray freezing (freezing) is typically accomplished by transporting a sample stage carrying the biological sample through a sample rod to a specific location in a freezing chamber, and then spraying the sample at high velocity with high pressure liquid nitrogen vapor. The high-pressure freezing (high pressure freeze) is similar to the plug-in freezing principle, adopts low-temperature liquid to freeze the sample, but applies about 2000 atmospheres of high pressure in the sample cavity while freezing, reduces the freezing temperature of water, and simultaneously inhibits the volume expansion generated in the ice crystallization process, thereby avoiding the damage of ice crystallization to the biological sample structure and preparing the frozen biological sample with higher quality.
However, insert freezing has the following drawbacks: because the whole sample needs to be inserted into the low-temperature liquid, the sample cannot be selectively frozen in a specific area in the freezing process, and real-time microscopic observation cannot be performed in situ in the freezing process. The injection type freezing is based on the insertion type 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 the high-pressure freezing has better freezing effect and higher sample quality because the high pressure inhibits ice crystallization. But spray freezing and high pressure freezing also have the disadvantage of not being able to be observed microscopically in real time and frozen in local selection. These drawbacks limit the further intensive research into freezing biological samples.
There is also provided in the prior art a device for rapid freezing of a sample, the device comprising: the sample container and the heating support device are arranged on the side face of the container to support the sample container, the sample container is placed on the base, and the quick freezing of the sample is realized by controlling the switch of the heating support device. The device has the advantages that the sample is arranged in the closed sample bearing structure, the wall of the sample bearing device separates the sample from the heating supporting device, and additional thermal resistance is generated, so that the freezing speed of the frozen sample is not ideal. In addition, in the aspect of heating and recovering frozen biological samples, the conventional method at present has a low heating speed, and auxiliary media such as DMSO (dimethyl sulfoxide) and the like are usually added into the samples to ensure that the biological samples are not damaged in the heating process, so that the activity of the biological samples is influenced, the biological samples such as cells and the like cannot be expressed, and the biological samples are truly expressed 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 contacts with a low-temperature cold source for freezing a sample, and the freezing chip comprises: the surface of the sample placement 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; a chip substrate supporting 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 is not overlapped or partially overlapped.
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 placing 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 apart locations of the localized temperature controlled region.
Optionally, when the chip substrate supports the top surface of the sample placing layer to form the first contact surface, the chip substrate is further provided with a second contact surface for contacting with the low-temperature cold source; the first contact surface and the second contact surface are positioned on the same side face of the chip substrate.
Optionally, the temperature control unit and the sample placement layer are an integrated structure.
Optionally, the temperature control unit is disposed on the sample placement layer by using 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 thermally conductive 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 alternatively
The sample placement layer includes: the chip micro-nano processing technology comprises a heat conduction layer and a first isolation layer which is manufactured on the heat conduction 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 first isolation layer; or alternatively
The sample placement layer includes: the device comprises a 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 alternatively
The sample placement layer includes: the device comprises a third isolation layer, a heat conduction layer, a first isolation layer and a second isolation layer, wherein 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 alternatively
The sample placement layer includes: the device comprises a third isolation layer, a first isolation layer, a heat conduction layer and a second isolation layer, wherein the first isolation layer is manufactured on the third isolation layer by adopting a chip micro-nano processing technology, the heat conduction layer is manufactured on the first isolation layer by adopting the 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; the temperature control unit is arranged on the third isolation layer so as to divide the local temperature control area on the second isolation layer.
Optionally, the sample placement layer comprises: at least one sample layer, a heating layer, a fourth isolation layer, a heat conduction layer and a fifth isolation layer which are arranged in a split manner; 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, a thickness of a portion of the heat conducting layer near the temperature control unit and an end portion of the heat conducting layer is greater than a thickness of a portion of the heat conducting layer therebetween; and/or 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 is arranged in a patterning structure.
Optionally, the local temperature controlled region is provided with at least one closed sample-receiving chamber and/or open sample-receiving chamber for receiving a sample.
Optionally, the temperature control unit further comprises an auxiliary temperature control unit provided 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 access channel to fit a microscope, a photodetector, an X-ray, a raman spectrometer, an infrared spectrometer.
Optionally, the frozen chip is made of a light-transmitting 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 frozen chip is controlled to be 0.1-2mm.
In a second aspect, embodiments of the present disclosure provide a sample stage assembly comprising 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: and the sample heat sink is used for accommodating the frozen chip.
In a third aspect, embodiments of the present disclosure provide a refrigeration system comprising a sample stage assembly according to any one of the second aspects. Specifically, the refrigeration system includes: a low-temperature cold source; and a heat sink base for fixing the sample stage assembly and contacting with the low-temperature cold source.
Optionally, the refrigeration system further comprises:
the freezing medium sealing cover plate is used for sealing the low-temperature cold source.
Optionally, the refrigeration system further comprises:
and the sample cover plate at least has an area capable of sealing the opening of the heat sink base.
In a fourth aspect, embodiments of the present disclosure provide a sample testing system comprising a refrigeration system according to the third aspect. Specifically, the sample testing system includes;
and a microscopic observation device and/or a detection device which are matched with the refrigeration system.
Optionally, the microscopic observation device is at least one of a positive optical microscope, an inverted optical microscope and an electron 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 the lowest temperature range which can be provided by the low-temperature cold source.
Optionally, the adjusting the electrical parameter of the temperature control unit to maintain the average temperature of the sample stable at the first temperature, and before maintaining the temperature gradient between the sample and the cryogenic cold source in the sample placement layer, the method further includes: adjusting the temperature of the local temperature control area to a first temperature; placing a sample within the localized temperature controlled region.
Optionally, the first temperature is changed to the second temperature for 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 enables the same sample to be directly converted into an amorphous solid state from a liquid state in the same environment, and keeps the temperature of the amorphous solid state continuously.
Optionally, the first temperature is 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 refrigeration 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 heating the sample by using an external heat source, and determining that the average temperature of the sample is at a first temperature by using a temperature measurement 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 period of time 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 converted from a liquid state to an amorphous solid state in the same environment and is continuously kept in the amorphous solid state.
Optionally, the first temperature is 0 ℃ to 40 ℃, and the second temperature is less than-140 ℃.
In a seventh aspect, embodiments of the present disclosure provide a method of manipulating 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 placing layer; and detecting and adjusting the electrical parameter to a first preset 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 in the lowest temperature range which can be provided by the cryogenic cold source.
Optionally, the method further comprises: adjusting an electrical parameter of a temperature control unit to a second predetermined range to heat the sample or to heat 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: and after the step of adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at a first temperature and the step of 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 the starting moment of adjusting the electrical parameters to a first preset range, and detecting and adjusting the electrical parameters to the first preset range at the starting moment so as 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 for a first predetermined period of time.
Optionally, the electrical parameters of the temperature control unit are adjusted by electronic means.
Optionally, the second temperature is changed to the third temperature for a second predetermined period of time.
Optionally, the second predetermined period of time 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 converted from a liquid state to an amorphous solid state in the same environment and is continuously kept in the amorphous solid state.
Optionally, the first temperature is 0 ℃ to 40 ℃, and the second temperature is less than-140 ℃.
Alternatively, the method is suitable for microscopic observation of a sample.
The technical scheme provided by the embodiment of the disclosure can comprise the following beneficial effects:
(1) According to the freezing chip disclosed by the embodiment of the disclosure, the temperature of the local temperature control area is adjusted by the temperature control unit through setting at least one local temperature control area, so that a sample can be selectively frozen, for samples which do not need to be frozen, the temperature control unit is controlled to release heat to maintain the temperature gradient of the sample and the low-temperature cold source, and for samples which need to be frozen, the electrical parameters of the temperature control unit are adjusted to enable the heat of the sample to be conducted to the low-temperature cold source, so that the effect of local selective freezing is realized.
(2) According to the freezing chip, the temperature control unit and the sample placement layer are integrally arranged, when the freezing chip is in contact with the low-temperature cold source, the temperature gradient of the sample and the low-temperature cold source is formed in the sample placement layer, and the sample heat can be quickly conducted along the direction of the temperature gradient by adjusting the electrical parameters of the temperature control unit, so that the quick freezing of the sample is realized, and low-temperature sample preparation such as a microscope, an X-ray device and the like can be provided for other testing devices.
(3) According to the refrigeration chip disclosed by the embodiment of the disclosure, through designing the structure of the heat conducting layer, the temperature gradient is limited to 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, so that the heat transfer speed is ensured, and meanwhile, the heat capacity of the refrigerated part is reduced, and the refrigeration speed is higher than 10 5 For cell samples, the quick freezing of the sample does not destroy the cell sample, facilitating a better study of the cell biological behavior.
(4) The freezing chip of the embodiment of the disclosure has the light passage channel on the sample placement layer, so that the sample can be subjected to in-situ characterization by the adaptation testing device, such as a microscope, an X-ray device and the like, thereby realizing the in-situ real-time sample testing while freezing the sample, and improving the sample testing efficiency.
(5) The sample testing system according to the embodiment of the present disclosure is used for operating the sample, and by adjusting the parameters of the temperature control unit, the operation procedure of freezing the sample-operation sample, or the cycle of freezing the sample-operation sample-heating the revival sample-freezing the sample-operation sample-heating the revival sample, or the operation procedure of pre-freezing the operation sample-freezing the sample, or the cycle of pre-freezing the operation sample-heating the revival sample-pre-freezing the operation sample-freezing the sample-operation sample-heating the revival sample can be realized, or the cycle of the above procedure can be repeated after freezing the sample-operation sample. According to the technical scheme, 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 are designed, so that the heat capacity of the local temperature control area is limited, and the temperature is higher than 10 5 The freezing and heating speed at the temperature of/s ensures that the structure and the function of the sample are not damaged in the repeated freezing and heating process, and the biological sample is frozen, observed in situ, thawed by heating and the likeThe operation is a great improvement, 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
In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the related art, a brief description will be given below of the drawings required for the exemplary embodiments or the related technical descriptions, and it is apparent that the drawings in the following description are some exemplary embodiments of the present disclosure, and other drawings may be obtained according to the drawings without inventive effort to those of ordinary skill in the art.
FIG. 1a shows a front view of a frozen chip according to an embodiment of the disclosure;
FIG. 1b shows a cross-sectional view in the direction DD' in FIG. 1;
FIG. 1c shows a cross-sectional view of a frozen chip according to another embodiment of the disclosure;
FIG. 1d shows a cross-sectional view of a frozen chip according to another embodiment of the disclosure;
FIGS. 2 a-2 e show schematic structural views of sample placement layers according to embodiments of the present disclosure;
FIG. 3 shows a schematic diagram of temperature gradients within a sample placement layer according to an embodiment of the present disclosure;
FIG. 4 shows a schematic diagram of a structure of a frozen chip on which a sample is placed according to an embodiment of the disclosure;
FIG. 5 illustrates a schematic structural view of a sample stage assembly according to an embodiment of the present disclosure;
FIG. 6 shows a schematic diagram of a refrigeration system according to an embodiment of the present disclosure;
FIG. 7 shows a flow diagram of a method of freezing a sample according to an embodiment of the present disclosure;
fig. 8 illustrates a basic principle schematic of the operation of a temperature control unit according to an embodiment of the present disclosure;
FIG. 9 shows a flow diagram of a method of heating a sample according to an embodiment of the present disclosure;
FIG. 10 shows a flow diagram of a method of microscopic observation of a sample in accordance with an embodiment of the present disclosure;
FIG. 11 shows a schematic of a chip and cell samples before and after on-chip freezing.
Fig. 12 shows a schematic of the freezing rate of the frozen chip according to fig. 2a-2 e.
Detailed Description
In order that those skilled in the art will better understand the present disclosure, a technical solution in exemplary embodiments of the present disclosure will be clearly and completely described in the following with reference to the accompanying drawings in exemplary embodiments of the present disclosure.
In some of the flows described in the specification and claims of this disclosure and in the foregoing figures, a number of operations are included that occur in a particular order, but it should be understood that the operations may be performed in other than the order in which they occur or in parallel, that the order of operations such as 101, 102, etc. is merely for distinguishing between the various operations, and that the order of execution does not itself represent any order of execution. In addition, the flows may include more or fewer operations, and the operations may be performed sequentially or in parallel. It should be noted that, the descriptions of "first" and "second" herein are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, and are not limited to the "first" and the "second" being different types.
Technical solutions in exemplary embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in exemplary embodiments of the present disclosure, and it is apparent that the described exemplary embodiments are only some embodiments of the present disclosure, not all embodiments. Based on the embodiments in this disclosure, all other embodiments that a person of ordinary skill in the art would obtain without making any inventive effort are within the scope of the disclosure.
In the prior art, the insert type freezing has the following defects: because the whole sample needs to be inserted into the low-temperature liquid, the sample cannot be selectively frozen in a specific area in the freezing process, and real-time microscopic observation cannot be performed in situ in the freezing process. The injection type freezing is based on the insertion type 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 the high-pressure freezing has better freezing effect and higher sample quality because the high pressure inhibits ice crystallization. But spray freezing and high pressure freezing also have the disadvantage of not being able to be observed microscopically in real time and frozen in local selection. These drawbacks limit the further intensive research into freezing biological samples. Meanwhile, no mature technology capable of recovering frozen samples by high-speed heating exists at present.
The present disclosure is provided to at least partially solve the problems in the prior art discovered by the inventors.
The three ways of freezing the chip and the insert freezing, the jet freezing and the high-pressure freezing provided by the disclosure are different in principle of freezing the sample. The difference is that the sample placed on the frozen chip is not in direct contact with the freezing medium (such as liquid nitrogen), but rather the sample is maintained at a higher temperature by means of external resistive heating while the freezing medium cools the sample stage (typically placing the chip on the sample stage, which 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, so that the sample is quickly frozen.
Fig. 1a shows a front view of a frozen chip according to an embodiment of the disclosure. The cryogenic heat sink a shown in fig. 1 b-1 d is not part of the frozen chip 10, and in the present disclosure, the cryogenic heat sink a is a device that provides a low temperature environment of the frozen chip 10 and is in direct contact with the frozen chip 10, for example, when the frozen chip 10 is used, is placed on a sample heat sink, and then the sample heat sink is fixed on a heat sink base immersed in the cryogenic heat sink, so that the sample heat sink also has the temperature of the cryogenic heat sink (such as liquid nitrogen) and can be regarded as the cryogenic heat sink a. The above is illustrative, and the present disclosure is not limited to the low temperature heat sink 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 to form 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 means of joule heating (electric current generates heat through a resistor), to adjust the temperature of the local temperature control zone N. The projection of the area P of the first contact surface 14 and the local temperature control area N on the same plane is not overlapped or partially overlapped.
According to the embodiment of the disclosure, the top surface of the sample placement layer 12 is used for placing a sample, the bottom surface of the chip substrate 11 usually supports the sample placement layer 12 to form a first contact surface 14 (as shown in fig. 1 b), the top surface of the chip substrate 11 may also support the sample placement layer 12 to form a first contact surface (as shown in fig. 1 c), fig. 1d also shows that the top surface of the chip substrate 11 supports the sample placement layer 12, unlike fig. 1c, when the top surface of the chip substrate 11 supports the sample placement layer 12 to form the first contact surface, the chip substrate 11 also has a second contact surface for contacting the cryogenic cold source a; the first contact surface and the second contact surface are positioned on the same side face of the chip substrate. According to embodiments of the present disclosure, the sample may be in direct contact with the sample placement layer 12, and may avoid creating additional thermal resistance and increase the freezing rate relative to non-direct contact.
The descriptions shown in fig. 1b-1d are used as schematic descriptions, and may be flexibly selected according to the needs, and the disclosure is not limited to the above arrangement, and will not be repeated herein.
According to embodiments of the present disclosure, the same plane may be the plane in which the sample placement layer 12 lies. 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 heat of the sample in the local temperature control region N is transferred from the local temperature control region N to the region P in the direction indicated by the arrow, and then transferred to the low-temperature heat sink a along the chip substrate 11. As shown in fig. 1c, the region P partially overlaps the local temperature control region N, and the heat of the sample in the local temperature control region N1 is transferred to the low-temperature cold source a along the chip substrate 11 after being transferred to the region P1 from the local temperature control region N1 in the direction indicated by the arrow, unlike fig. 1b, the heat of the sample in the local temperature control region N2 is transferred to the low-temperature cold source a along the chip substrate 11 after being transferred to the temperature control unit 13 from the local temperature control region N2 in the direction indicated by the arrow.
It should be noted that, the wires of the temperature control unit 13 may penetrate through the sample placement layer 12 and be connected to the controller outside the frozen chip 10, and the heat generated by the wire portion may be negligible when the temperature of the sample in the local temperature control region N is adjusted.
The frozen chip shown in fig. 1b-1d, the heat of the sample can be conducted to the cryogenic cold source a in the direction of transverse conduction and longitudinal conduction, thereby freezing the sample. Moreover, the central region of the chip substrate 11 is hollow, and may be adapted for in situ characterization of a sample by a testing device, such as a microscope, X-ray device, etc., without limitation of the present disclosure.
When the frozen chip provided by the disclosure is used, before freezing a sample, the frozen chip is placed on the low-temperature cold source A, the temperature control unit 13 maintains the sample at a first temperature, for example, 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, and the heat of the sample in the local temperature control area N is conducted along the temperature gradient direction, so that the rapid freezing of the sample is realized, and then the temperature of the sample is adjusted to the required second temperature by detecting the electrical parameters, for example, when the low-temperature cold source A can provide the low temperature of-190 ℃, the temperature of the sample can be adjusted to-140 ℃.
The second temperature is determined according to the temperature of the low-temperature cold source a, and may be not lower than the second temperature, which is not limited in the present disclosure.
According to the freezing chip disclosed by the embodiment of the disclosure, the temperature of the local temperature control area is adjusted by the temperature control unit through setting at least one local temperature control area, so that a sample can be selectively frozen, for samples which do not need to be frozen, the temperature control unit is controlled to release heat to maintain the temperature gradient of the sample and the low-temperature cold source, and for samples which need to be frozen, the electrical parameters of the temperature control unit are adjusted to enable the heat of the sample to be conducted to the low-temperature cold source, so that the effect of local selective freezing is realized.
According to an embodiment of the present disclosure, the chip substrate 11 is supported at a 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 localized temperature controlled region. For example, the chip substrate 11 is a surrounding structure adapted to the periphery outside the central area 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 and is supported on one side or two sides of the central area; wherein the central region is divided into at least one local temperature control region N. The central region may be adapted above or below to allow in situ characterization of the sample by a testing device, such as a microscope, X-ray device, etc.
As another embodiment, the chip substrate 11 is supported on the central region of the sample placement 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 placement layer 12 is disposed horizontally, and 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 periphery of the support region.
In some cases, the chip substrate 11 may also be supported at spaced apart positions 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, so that the local temperature control area N can be divided in the area between the supporting blocks and the peripheral area outside the supporting blocks.
According to the embodiment of the present disclosure, the frozen chip 10 is manufactured by a chip micro-nano processing process, such as a thin film deposition process, a dry or wet etching process, a photolithography process, etc. in the chip field, which will not be described herein.
According to the embodiment of the disclosure, the overall thickness of the frozen chip 10 is controlled to be 0.1-2mm.
According to the embodiment of the disclosure, the sample placement layer 12 is provided with an optical path channel, so that the sample can be subjected to in-situ characterization by a testing device, such as a microscope, an X-ray device and the like, thereby realizing the in-situ real-time testing of the sample while freezing the sample, and improving the sample testing efficiency. Specifically, the freezing chip is made of a light-transmitting material or has a perforation channel as the light passage channel to be adapted to monitoring instruments such as an upright optical microscope, an inverted optical microscope, an electron microscope, a photodetector, an X-ray, a raman spectrometer, an infrared spectrometer, and the like.
According to an embodiment of the present disclosure, the chip substrate 11 is used as a mechanical carrier part of the frozen chip 10, and the thickness of the chip substrate 11 is typically 0.1-2mm, and the material used is typically silicon (such as silicon wafer) or silicon carbide.
According to the embodiment of the disclosure, the temperature control unit 13 is disposed in the sample placement layer 12 by using a chip micro-nano processing technology, and the local temperature control area N is divided by using the temperature control unit 13. Each local temperature control region N may be independently controlled by the corresponding temperature control unit 13 to heat 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 may be adjusted together in combination, which is not limited in the present disclosure.
According to an embodiment of the present disclosure, the thickness of the temperature control unit 13 is typically 0.1-5um, and the material used is typically 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 diagrams of a sample placement layer according to embodiments of the present disclosure. As shown in fig. 2a to 2e, the sample placement layer 12 includes: a heat conductive layer 121, a first insulating layer 122, a second insulating layer 123, and a third insulating layer 124. The heat conducting layer 121 is used for transversely conducting heat of a sample to the low-temperature cold source A, the first isolating layer 122 is used for isolating the heat conducting layer 121 and the temperature control unit 13, the second isolating layer 123 is used for isolating the temperature control unit 13 from the external contact environment, the temperature control unit 13 is insulated and protected, and the third isolating layer 124 is used for isolating the chip substrate 11 and the heat conducting layer 121. The first isolation layer 122, the second isolation layer 123, and the third isolation layer 124 may be omitted according to circumstances.
The material of the heat conducting layer 121 may be metal (such as aluminum, copper, platinum, etc.), heat conducting ceramic (such as alumina, aluminum nitride, etc.), or other heat conducting materials (such as silicon, silicon carbide, silicon nitride, etc.). The thickness of the thermally conductive layer 121 is typically 0.1-5um.
According to an embodiment of the present disclosure, the temperature control unit 13 is of an integrated structure with the sample placement layer 12.
As shown in fig. 2a, the sample placement layer 12 includes only the heat conductive layer 121; the temperature control unit 13 is disposed on the heat conductive layer 121 to divide the local temperature control region N on the heat conductive layer 121. In this embodiment, the sample placement layer 12 is composed of only a heat conductive layer, and the power consumption for maintaining the temperature of the sample is large, but the freezing speed of the freezing chip can reach 10 5 -10 6 ℃/s。
As shown in fig. 2b, the sample placement layer 12 includes: a heat conducting layer 121 and a first isolation layer 122 manufactured on the heat conducting 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 area on the first isolation layer 122. In this embodiment, the freezing speed of the frozen chip is small compared to fig. 2a, and can still reach 10 5 -10 6 ℃/s。
As shown in fig. 2c, the sample placement layer 12 includes: the semiconductor device comprises a heat conduction layer 121, a first isolation layer 122 manufactured on the heat conduction layer 121 by adopting a chip micromachining process and a 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 that the temperature control unit 13 is prevented from being exposed to the external environment, and the service life of the frozen chip is prolonged, and the freezing speed of the frozen chip can still reach 10 through testing 5 -10 6 ℃/s。
As shown in fig. 2d, the sample placement layer 12 includes: a third isolation layer 124, a heat conduction layer 121 formed on the third isolation layer 124 by chip micro-nano processing technology, and a chip micro-nano processing technologyA first isolation layer 122 formed on the heat conductive layer 121 by a nano-processing process and a second isolation layer 123 formed on the first isolation layer 122 by a chip micro-nano-processing 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 third isolation layer 123 is disposed under the heat conducting layer 121, and considering that the heat conducting layer 121 is usually made of a metal material, the third isolation layer 123 can be disposed between the heat conducting layer 121 and the chip substrate 11 based on convenience of a processing process, so as to meet process requirements, and the freezing speed of the frozen chip can still reach 10 after testing 5 ℃/s。
As shown in fig. 2e, the sample placement layer 12 includes: the semiconductor device 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, unlike 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 high, and the freezing speed of the frozen chip can still reach 10 after testing 5 ℃/s。
Specifically, as shown in FIG. 12, for the blank chip, the temperature was 1.2ms when the temperature was lowered from 300K (corresponding to the horizontal axis time point of 1.4 ms) to 90K (corresponding to the horizontal axis time point of 2.6 ms), and the freezing rate reached about 1.8X10 5 In the same way, the water-containing chip for freezing the sample only needs 2.2ms to freeze the sample temperature from 300K (corresponding to the time point of 1.4ms on the transverse axis) to 90K (corresponding to the time point of 3.6ms on the transverse axis), and the freezing rate reaches 1.0x10 5 DEG C/S. In the present disclosure, a blank chip refers to a chip that is not loaded with a sample, and an aqueous chip refers to a chip that is loaded with a liquid sample, unless otherwise specified.
In fig. 2 a-2 e, the heat conducting layer 121 is preferably made of a material with high thermal conductivity, such as a metal material, to increase the freezing speed.
The specific manner shown above is used as a schematic illustration, and may be flexibly selected according to needs, and the disclosure is not limited to the above manner, and will not be repeated here.
According to the refrigeration chip disclosed by the embodiment of the disclosure, by designing the structure of the heat conducting layer, the temperature gradient is limited to 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, so that the heat capacity of a local temperature control area is limited, and the refrigeration speed is higher than 10 5 For cell samples, the quick freezing of the sample does not destroy the cell sample, facilitating a better study of the cell biological behavior.
It will be appreciated by those of ordinary skill in the art that, depending on the design requirements, the heat conductive layer, the first isolation layer, and the second isolation layer in the chip substrate, the sample placement layer, and the sample placement layer may be discontinuous, and holes, slots, etc. may be formed therein to adjust the thermal conductivity or facilitate light transmission observation.
As another embodiment, the sample placement layer 12 includes: at least one sample layer, a heating layer, a fourth isolation layer, a heat conduction layer and a fifth isolation layer which are arranged in a split manner; 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.
Unlike the sample placement layer shown in fig. 2a to 2e, the sample placement layer 12 has a non-integrated structure as a whole, and when in use, the sample layer, the heating layer, the fourth isolation layer, the heat conduction layer, and the fifth isolation layer are sequentially stacked and fixed by an external fixture. The sample layer and other layers are independently arranged, the heating layer, the fourth isolation layer, the heat conduction layer and the fifth isolation layer can be independently arranged, and two or three layers of the sample layer can be combined by adopting a chip micro-nano processing technology, and the sample layer and the other layers are combined according to the superposition sequence when the sample layer is placed for use. The sample layers can be independently arranged, so that the number of the sample layers can be flexibly set according to the requirement, and the sample layers can be replaced in time when a certain sample layer is damaged. Sample in split arrangementCompared with the sample placement layer with an integrated structure, the placement layer can generate new thermal resistance between layers, and generally affects the freezing speed of the frozen chip. When the heat of the frozen chip provided by the embodiment of the disclosure is transmitted 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 the freezing speed can be 10 through tests 5 On the order of DEG C/s.
It should be noted that, for further technical details of the sample layer, the heating layer, the fourth isolation layer, the heat conducting layer and the fifth isolation layer, reference may be made 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 isolation layer corresponds to the first isolation layer and is used for isolating the temperature control unit and the heat conducting layer; the fifth isolation layer corresponds to the third isolation layer, and is used for isolating the chip substrate and the heat conducting layer, and is not described herein.
In addition, the frozen chip provided by the present disclosure may be further improved in several aspects:
a, adjusting the thickness of the heat conducting layer part, wherein the thickness of the part close to the temperature control unit and the thickness of the end part of the heat conducting layer are larger than the thickness of the heat conducting layer part between the two parts;
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 pattern structure, for example, the part is connected with 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 a schematic diagram of a temperature gradient within a sample placement layer according to an embodiment of the present disclosure. As shown in FIG. 3, the temperature of the low temperature heat sink A is-170℃and the temperature at the point w1 at the bottom of the chip substrate 11 is approximately-160℃as the temperature of the low temperature heat sink A. The temperature at the point w2 at the top of the sample placement layer 12 is, for example, -120 ℃. The temperature near the point w3 of the temperature control unit 13, which is located on the same plane as the position point w2, 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 not limiting 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 reduced as much as possible, for example, the range of the local temperature control region is made small enough, and the temperature control unit is close to the sample as much as possible, so that the heat capacity of the local temperature control region is limited, and the freezing speed can be improved. On the other hand, the temperature gradient is concentrated in the area close to the temperature control unit as much as possible by adopting a structure with lower relative heat conductivity at the position close to the temperature control unit outside the temperature control unit, for example, the temperature gradient is concentrated between the point w3 and the point w4 instead of between the point w3 and the point w2, so as to improve the freezing speed. The improvement of the two aspects is combined, which is beneficial to improving the freezing speed.
By adopting the mode a and/or the mode b to improve the freezing chip, the freezing speed of the freezing chip can be further provided, and the freezing speed can be 10 after test 5 On the order of DEG C/s.
Fig. 4 shows a schematic structural view of a frozen chip on which a sample is placed according to an embodiment of the present disclosure. As shown in fig. 4, the local temperature controlled region is provided with at least one closed sample receiving chamber a and/or open sample receiving chamber b for receiving a sample, unlike fig. 1 a. Of course, closed sample receiving chambers a and/or open sample receiving chambers b may also be provided on the basis of the frozen chips shown in FIGS. 1b-1c, as this disclosure is not limited in this regard. For other technical details of the frozen chip according to the embodiments of the present disclosure, refer to the embodiment parts shown in fig. 1a to 1c, and are not described herein.
According to an embodiment of the present disclosure, the temperature control unit 12 further comprises an auxiliary temperature control unit provided on the wall of the closed sample receiving chamber a and/or the open sample receiving chamber b for reducing the temperature difference between a plurality of samples placed in the same local temperature control area. In the present embodiment, the auxiliary temperature control unit and the temperature control unit may use the same component or equivalent components.
Fig. 5 shows a schematic structural view of a sample stage assembly according to an embodiment of the present disclosure. As shown in fig. 5, the sample stage assembly 20 includes: a frozen chip 10, a sample heat sink 21 and a controller 22. Wherein the sample heat sink 21 is used for accommodating the frozen chip 10. The controller 22 is electrically connected to the temperature control unit 13, and is used for adjusting the temperature of the temperature control unit 13. It should be noted that the sample heat sink 21 may be designed as a light-transmitting structure to fit the microscope to observe the sample.
In the disclosed manner, the sample heat sink 21 in the sample stage assembly 20 may be considered a cryogenic cold source a. It will be appreciated that the sample heatsink 21 may be omitted and the frozen chip 10 may be placed directly on the heatsink base 32 described below, at which time the heatsink base 32 may be considered a cryogenic 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 drawings), which may be embedded in the sample heat sink 21 or disposed around a region where the sample heat sink 21 is in direct contact with the freezing chip 10, so as not to affect efficient heat transfer of both, and the present disclosure is not limited to the position of the control circuit board. The controller 22 is electrically connected with the temperature control unit 13 through a control circuit board, so as to adjust the temperature of the temperature control unit 13.
Fig. 6 illustrates a structural schematic of a refrigeration system according to an embodiment of the present disclosure. As shown in fig. 6, the refrigeration system 30 includes: sample stage assembly 20, cryogenic heat sink 31 and heatsink base 32. The cryogenic cold source 31 may be liquid nitrogen for cooling and maintaining the heatsink base 32 close to the temperature of the liquid nitrogen. The heat sink base 32 is used to secure the sample stage assembly 20 and to freeze the sample stage assembly 20 as a cold source.
In accordance with embodiments of the present disclosure, when freezing a sample, the heat sink base 32 is in direct contact with the sample heat sink 21 such that the temperature of the sample heat sink 21 is close to or the same as the liquid nitrogen temperature, and other portions of the sample stage assembly 20 except for the local temperature control region 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, the ambient temperature of which 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 includes: 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 also be supported to be immersed in the low-temperature cold source.
According to an embodiment of the present disclosure, the refrigeration system 30 further includes: a sample cover plate 34 having an area capable of sealing at least the opening of the heatsink base 32. The lengths of the sample cover plates 34 shown in the figure extend to two ends of the freezing medium sealing cover plate 33 respectively, so that the situation that water vapor does not enter in the low-temperature environment where the freezing chip is located is ensured, liquid drops formed by condensation of the water vapor are prevented from being attached to the sample, and further microscopic observation or property characterization of the sample is prevented from being influenced by ice crystals formed by the liquid drops in the low-temperature environment. It will be appreciated that the sample cover 34 is of sufficient area to cover the sample heat sink, and is typically capable of sealing the cryogenic environment in which the frozen chip is located from moisture ingress, and on this basis, the length of the sample cover 34 may be suitably increased, as this disclosure is not limited in this regard.
In the disclosed manner, the sample cover 34 may also be provided with an observation or detection zone to microscopically observe the sample through the observation zone and/or characterize the sample's properties at the detection zone location with a detection device, provided that moisture ingress is prevented in a low temperature environment. In some cases, a dry atmosphere may be provided for a low temperature environment to address the defect that moisture condensation affects sample observation or characterization, at which point the sample cover 34 may be omitted.
The present disclosure also provides a sample testing system comprising a refrigeration system 30 and a microscopic observation device and/or detection device for use with the refrigeration 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, an infrared spectrometer and the like.
Fig. 7 shows a 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 the refrigeration system 30 to freeze a sample, including the following steps S110-S140.
In step S110, the temperature of the local temperature control region is adjusted to a first temperature;
in the disclosed mode, first, a control circuit board is connected with a controller in a room temperature state; secondly, the controller is started to heat the temperature control unit to a set temperature slightly higher than the room temperature (the temperature of the temperature control unit is determined by measuring the resistance value in real time, such as 30 ℃), and the temperature is kept constant (the temperature is adjusted by resistance feedback), and the temperature of the sample can be approximately considered to be also at the set temperature (such as 30 ℃) because the distance between the temperature control unit and the sample is extremely small and the thermal resistance is extremely low, and the typical resistance value at the moment is in the range of rheter=50-100 ohms.
In step S120, placing a sample within the localized temperature controlled region;
in step S130, adjusting an electrical parameter of the temperature control unit to maintain the average temperature of the sample stable at the first temperature, and maintaining 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 frozen heat sink base (about-190 ℃), the temperature of the frozen chip begins to decrease, at this time, the controller automatically increases the current Iheater to perform resistance heating, so that the average temperature of the sample in the local temperature control area N is maintained at a first temperature (for example, 30 ℃), at this time, the typical current value ranges from iheater=50 to 100ma, and the typical power of rheter (rheter×iheater 2) is about 0.3W;
in step S140, the electrical parameter is detected and adjusted to a first predetermined range, so as 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 cryogenic cold source.
In the disclosed manner, when freezing is needed, a signal is sent out by the controller to suddenly reduce the current Iheater 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, and rheter is also rapidly reduced to about 1/7 of rheter at room temperature, and the control circuit maintains a small constant current (0.1-1.0 mA) in the whole cooling process. After the freezing is finished, the control circuit keeps a small current (0.1-1.0 mA), the average temperature of the sample is maintained at a second temperature (such as-190 ℃), and the Rheater change is continuously monitored and used as a reference of the temperature of the sample.
In the embodiment of the present disclosure, the second temperature is determined according to the temperature of the low-temperature heat sink a, and may be not lower than the temperature. Specifically, 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, -140 ℃.
It should be noted that, step S110 and step 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 be room temperature, and 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 following describes the basic principle of the operation of the temperature control unit:
fig. 8 illustrates a basic principle schematic 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 in a 4-terminal measurement manner, namely force_H (I+), sense_H (V+), sense_L (V-), force_L (I-). The heating current Iheater is applied through I+ to I-, and can reach the maximum of 50-200 mA. The voltage difference Vheater is measured at both the V+ and V-terminals, and the port current at the two terminals is small (e.g., virtual ground), and the influence of the current passing through the temperature control unit is not recorded. The resistance value rheter of the temperature control unit was measured in real time by Vheater/Iheater, and the average temperature of the temperature control unit was evaluated in this manner.
It should be noted that, in the embodiment of the disclosure, the function of local area selection freezing can be achieved by controlling the temperature control units corresponding to different local temperature control areas, the temperature control units and the local temperature control areas can be in a one-to-one correspondence relationship, and of course, the temperature of a plurality of local temperature control areas can be adjusted by adopting one temperature control unit according to the need, so that a person skilled in the art can freely combine the temperature control units, and the function of rapidly freezing samples can be achieved by adopting the above-mentioned mode. The present disclosure is not limited in this regard.
According to embodiments of the present disclosure, the average temperature of the sample is adjusted by adjusting the electrical parameter. Wherein the electrical parameter may be a current, resistance, or power parameter, which is not limited by the present disclosure.
In the disclosed mode, the temperature of the sample can be measured in real time while the sample is heated by using the temperature control unit, and a temperature measuring unit can be additionally arranged on the freezing chip, the sample is heated by using the temperature control unit, and the temperature of the sample is measured in real time by using the temperature measuring unit. The present disclosure is not limited in this regard.
In the disclosed manner, a change in resistance over time may be plotted and then the sample cooling rate is estimated from the change in resistance over time. Specifically, under the condition that the Iheater current is kept unchanged, the Rheater is calculated by measuring the Vheater, and a change curve of the Rheater along with time in the cooling process is continuously monitored, wherein the curve can be used as an evaluation reference of the sample freezing speed.
According to an embodiment of the present disclosure, the first temperature is changed to the second temperature for a predetermined period of time.
In the disclosed manner, the predetermined period of time for which the first temperature is reduced to the second temperature is controlled to be within 10ms, for example, 1-2ms. Specifically, the temperature is reduced from room temperature to below-140 ℃ within 1ms, and further reduced to below-180 ℃ within the subsequent 1-2ms.
According to embodiments of the present disclosure, the time delay may be a delay time for the control system to begin freezing the sample after sending an electrical signal that reduces the first temperature until the electrical signal is received by the freezing chip. It will be appreciated that when testing biological samples, it is necessary to determine the point in time at which the biological sample is frozen, to observe the sample at that point in time or to conduct other tests. The time delay reflects the delay time of the freezing operation, and the smaller the time delay is, the more accurate 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 in the freezing operation, and the sample test is better performed.
According to the embodiment of the present disclosure, by optimizing the circuit structure and the control method of the temperature control unit, the time delay can be controlled to be less than 0.1ms.
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, for a conventional cell sample, the temperature is in the range of 0-40 ℃, preferably 20-30 ℃; for specific heat resistant cells or bacteria, the temperature may be increased; under the condition of non-normal pressure, the temperature range can also be changed so as to ensure that the culture solution is in a liquid state and the biological sample normally survives.
According to the embodiment of the disclosure, the second temperature is a temperature at which the same sample is directly converted 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 the temperature range may be changed at high or low pressure 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 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 refrigeration system 30 to heat the sample, including the following steps S210-S220.
In step S210, the electrical parameter is detected and adjusted to bring the average temperature of the local temperature-controlled region to the second temperature.
In the disclosed mode, first, a temperature control unit is connected with a controller under a low-temperature (liquid nitrogen temperature) condition; secondly, a control circuit is started, the set value of the I_Heater is 0.1-1.0mA (only the resistance value is measured 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 heating the sample by using an external heat source, and determining that the average temperature of the sample is at a first temperature by using a temperature measurement 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, IHeater is suddenly increased, and the Rheater is heated to a Rheater value corresponding to a set temperature (such as 30 ℃) at the fastest speed. In the process, the initial Rheater is the resistance value at the temperature of liquid nitrogen and is only about 1/7 of the room temperature, so that the initial heating current reaches 200-300 mA level and reaches 0.3W equivalent power, thereby achieving the purpose of rapid heating. Meanwhile, during the heating process, the resistance value is rapidly increased, so that the Iheater needs to be rapidly adjusted (reduced) to a reasonable range, thereby maintaining the rheter at a set value (such as rheter corresponding to 30 ℃). The heating element is then maintained stable at a set temperature (e.g., 30℃.) and the sample can be removed or frozen as desired.
In the disclosed mode, the external heat source can be utilized to limit the heating area to the local temperature control area on the freezing chip through focusing, so as to heat the sample, then the control of heating power and temperature is realized through matching with the feedback system on the freezing chip, for example, the temperature measuring unit can be arranged on the freezing chip to monitor the temperature of the sample in real time, and then the heating power of the external heat source is controlled. Wherein the external heat source can be microwave, laser, etc.
After the sample is frozen using the frozen chip, step S210 may be omitted, and step S220 may be directly performed to heat the sample.
The method for heating a sample according to the embodiments of the present disclosure uses the refrigeration system 30 to heat the sample, and specific technical details refer to the embodiment shown in fig. 6, which is not described 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, such as 1-2ms.
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, for a conventional cell sample, the temperature is in the range of 0-40 ℃, preferably 20-30 ℃; for specific heat resistant cells or bacteria, the temperature may be increased; under the condition of non-normal pressure, the temperature range can also be changed so as to ensure that the culture solution is in a liquid state and the biological sample normally survives.
According to the embodiment of the disclosure, the second temperature is a temperature at which the same sample is directly converted 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 the temperature range may be changed at high or low pressure 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. 10 shows a flow diagram of a method of manipulating a sample according to an embodiment of the present disclosure. As shown in fig. 10, the method uses 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 maintaining a temperature gradient between the sample and the low-temperature cold source in the sample placement layer;
in step S320, the electrical parameter is detected and adjusted to a first predetermined range, so as 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 a required temperature value is determined within the lowest temperature range that can be provided by the cryogenic cold source;
in step S330, adjusting an electrical parameter of a temperature control unit to a second predetermined range to heat the sample or to heat 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;
in step S340, the sample is replaced after the sample is handled.
It should be noted that, step S340 may be performed after the sample is heated to the first temperature in step S320, that is, after the sample is once operated at the second temperature, the sample may be repeatedly frozen according to need after the sample is heated to the first temperature, and the operation is finished after the sample is operated for the second time, which is not limited by the cycle number of freezing, heating, and refreezing in the present disclosure. It will be appreciated that after the operation is completed in step S320, a new sample may also be replaced at the first temperature and then the new sample is repeatedly frozen, which is not limited by the present disclosure.
The specific technical details of the method for operating a sample provided in the embodiments of the present disclosure refer to the embodiments shown in fig. 7 and 9, and the embodiments of the present disclosure are not described herein.
According to an embodiment of the present disclosure, the operation sample may be a microscopic observation sample, a detection signal of a test sample under a monitoring instrument such as a photodetector, an X-ray, a raman spectrometer, an infrared spectrometer, and the like, which is not limited in this disclosure.
According to an embodiment of the present 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 maintain the temperature gradient between the sample and the cryogenic cold source in the sample placement layer in step S310, the method further includes:
Operating the sample at a first temperature and determining a start time to adjust the electrical parameter to a first predetermined range, and detecting and adjusting the electrical parameter to the first predetermined range at the start time 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 for a first predetermined period of time.
According to an embodiment of the disclosure, the electrical parameters of the temperature control unit are adjusted by an electronic device. The time delay may be controlled to within 2ms, for example, by adjusting the electrical parameters of the temperature control unit using keithley 2612B.
According to the embodiment of the present disclosure, by optimizing the circuit structure and the control method of the temperature control unit, the time delay can be controlled to be less than 0.1ms.
According to an embodiment of the present disclosure, the second temperature is changed to the first temperature for a second predetermined period of time.
According to an embodiment of the present disclosure, the second predetermined period of time is within 10ms, for example 1-2ms.
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, for a conventional cell sample, the temperature is in the range of 0-40 ℃, preferably 20-30 ℃; for specific heat resistant cells or bacteria, the temperature may be increased; under the condition of non-normal pressure, the temperature range can also be changed so as to ensure that the culture solution is in a liquid state and the biological sample normally survives.
According to the embodiment of the disclosure, the second temperature is a temperature at which the same sample is directly converted 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 the temperature range may be changed at high or low pressure 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.
The sample testing system according to the embodiment of the present disclosure is used for operating the sample, and by adjusting the parameters of the temperature control unit, the operation procedure of freezing the sample-operation sample, or the cycle of freezing the sample-operation sample-heating the revival sample-freezing the sample-operation sample-heating the revival sample, or the operation procedure of pre-freezing the operation sample-freezing the sample, or the cycle of pre-freezing the operation sample-heating the revival sample-pre-freezing the operation sample-freezing the sample-operation sample-heating the revival sample can be realized, or the cycle of the above procedure can be repeated after freezing the sample-operation sample. According to the technical scheme, 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 are designed, so that the heat capacity of the local temperature control area is limited, and the temperature is higher than 10 5 The freezing and heating speed at the temperature of/s ensures that the sample is kept from being damaged (or the damage is lightened) in the repeated freezing and heating process, which is a great improvement for the operations of biological sample freezing, in-situ observation, heating and thawing and the like, and has great significance and wide application prospect.
The manner in which a sample is microscopically viewed using the sample testing system provided by embodiments of the present disclosure is described in detail below.
Placing a sample in a local temperature control area, keeping the sample at a first temperature, freezing the sample to a second temperature, and carrying out microscopic observation, wherein the method is suitable for carrying out high-resolution microscopic observation on a protein sample after freezing and preparing the sample;
mode two: the method is suitable for cell samples, and can be used for observing the activity of the sample in real time, freezing the sample at a specific time point of interest, such as cell division and foreign matter phagocytosis, 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 may be different, so that observation with different resolutions is achieved. For example, a sample is observed in real time by using a conventional upright optical microscope, and after freezing, the cells are observed in a high-resolution structure by using an electron microscope.
The method for microscopic observation of the sample provided by the embodiment of the disclosure comprises the steps of freezing a cell sample from 20-30 ℃ to about-170 ℃ for less than 2ms, wherein the freezing speed is higher than 10 5 The temperature/s is higher than that of the cell sample, so that the cell sample is basically unchanged in shape after freezing, and the cell sample is not broken and obviously deformed. The foregoing description is only of the preferred embodiments of the present disclosure and description of the principles of the technology being employed. It will be appreciated by those skilled in the art that the scope of the invention referred to in this disclosure is not limited to the specific combination of features described above, but encompasses other embodiments in which any combination of features described above or their equivalents is contemplated without departing from the inventive concepts described. Such as those described above, are mutually substituted with the technical features having similar functions disclosed in the present disclosure (but not limited thereto).

Claims (45)

  1. A frozen chip, characterized in that the frozen chip is in contact with a cryogenic cold source for freezing a sample, comprising:
    the surface of the sample placement 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;
    A chip substrate supporting 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 is not overlapped or partially overlapped.
  2. The frozen chip as recited in claim 1, wherein,
    the chip substrate is supported in a peripheral area outside the central area of the sample placement layer; the central area is divided into at least one local temperature control area; or alternatively
    The chip substrate is supported in the central area of the sample placement layer; the peripheral area outside the central area is divided into at least one local temperature control area; or alternatively
    The chip substrate is supported at spaced apart locations of the localized temperature controlled region.
  3. The frozen chip as recited in claim 1, wherein,
    when the chip substrate supports the top surface of the sample placing layer to form the first contact surface, the chip substrate is also provided with a second contact surface for contacting with the low-temperature cold source; the first contact surface and the second contact surface are positioned on the same side face of the chip substrate.
  4. The frozen chip as recited in claim 1, wherein,
    the temperature control unit and the sample placement layer are of an integrated structure.
  5. The frozen chip of claim 1, wherein the temperature control unit is disposed on the sample placement layer using a chip micro-nano process, and the temperature control unit is utilized to divide the local temperature control region.
  6. The frozen chip as recited in claim 5, wherein,
    the sample placing 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 alternatively
    The sample placement layer includes: the chip micro-nano processing technology comprises a heat conduction layer and a first isolation layer which is manufactured on the heat conduction 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 first isolation layer; or alternatively
    The sample placement layer includes: the device comprises a 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 alternatively
    The sample placement layer includes: the device comprises a third isolation layer, a heat conduction layer, a first isolation layer and a second isolation layer, wherein 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 alternatively
    The sample placement layer includes: the device comprises a third isolation layer, a first isolation layer, a heat conduction layer and a second isolation layer, wherein the first isolation layer is manufactured on the third isolation layer by adopting a chip micro-nano processing technology, the heat conduction layer is manufactured on the first isolation layer by adopting the 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; the temperature control unit is arranged on the third isolation layer so as to divide the local temperature control area on the second isolation layer.
  7. The frozen chip as recited in claim 1, wherein,
    the sample placement layer includes: at least one sample layer, a heating layer, a fourth isolation layer, a heat conduction layer and a fifth isolation layer which are arranged in a split manner;
    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.
  8. The frozen chip according to claim 6 or 7, characterized in that the thickness of the portion of the heat conducting layer close to the temperature control unit and the end portion of the heat conducting layer is greater than the thickness of the heat conducting layer portion therebetween; and/or
    The part of the heat conduction layer, which is close to the temperature control unit, and the part of the heat conduction layer, which is between the end part of the heat conduction layer, are arranged in a patterning structure.
  9. A frozen chip according to any of claims 6-8, characterized in that the local temperature controlled area is provided with at least one closed sample receiving cavity and/or open sample receiving cavity for receiving a sample.
  10. The frozen chip according to claim 9, characterized in that the temperature control unit further comprises an auxiliary temperature control unit provided on the wall of the closed sample receiving cavity and/or open sample receiving cavity.
  11. The frozen chip according to any one of claims 1-10, characterized in that the sample placement layer is provided with a light path channel to fit a microscope, a photodetector, an X-ray, a raman spectrometer, an infrared spectrometer.
  12. The frozen chip according to claim 11, which is made of a light-transmitting material or has a perforated channel as the light passage channel.
  13. The frozen chip according to any one of claims 1-12, characterized in that the frozen chip is manufactured with a chip micro-nano process.
  14. The frozen chip according to claim 13, characterized in that the thickness of the frozen chip is controlled between 0.1 and 2mm.
  15. A sample stage assembly comprising a frozen chip according to any one of claims 1-14, comprising:
    And the controller is electrically connected with the temperature control unit and is used for adjusting the temperature of the temperature control unit.
  16. The sample stage assembly of claim 15, further comprising: and the sample heat sink is used for accommodating the frozen chip.
  17. A refrigeration system comprising the sample stage assembly of claim 15 or 16, comprising:
    a low-temperature cold source;
    and a heat sink base for fixing the sample stage assembly and contacting with the low-temperature cold source.
  18. The refrigeration system of claim 17, further comprising:
    the freezing medium sealing cover plate is used for sealing the low-temperature cold source.
  19. The refrigeration system of claim 17, further comprising:
    and the sample cover plate at least has an area capable of sealing the opening of the heat sink base.
  20. A sample testing system comprising a refrigeration system according to any one of claims 17 to 19, comprising;
    and a microscopic observation device and/or a detection device which are matched with the refrigeration system.
  21. The sample testing system of claim 20, wherein said microscopic viewing device is at least one of an upright optical microscope, an inverted optical microscope, an electron microscope;
    The detection device is at least one of a photoelectric detector, an X-ray, a Raman spectrometer and an infrared spectrometer.
  22. A method for freezing a sample using a freezing system according to any one of claims 17 to 19, 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;
    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 the lowest temperature range which can be provided by the low-temperature cold source.
  23. The method of claim 22, wherein the adjusting the electrical parameter of the temperature control unit to maintain the average temperature of the sample stable at the first temperature, the method further comprises, prior to maintaining the temperature gradient between the sample and the cryogenic cold source within the sample placement layer:
    adjusting the temperature of the local temperature control area to the first temperature;
    placing a sample within the localized temperature controlled region.
  24. The method of claim 22, wherein the step of determining the position of the probe is performed,
    Changing the first temperature to the second temperature over a predetermined period of time.
  25. The method of claim 24, wherein the predetermined period of time is within 10 ms.
  26. The method of claim 22, wherein the electrical parameters of the temperature control unit are adjusted by an electronic device.
  27. The method of claim 22, wherein the step of determining the position of the probe is performed,
    the first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly converted from the liquid state to the amorphous solid state in the same environment and the amorphous solid state is continuously maintained.
  28. The method of claim 27, wherein the step of determining the position of the probe is performed,
    the first temperature is 0 ℃ to 40 ℃ and the second temperature is less than-140 ℃.
  29. A method for heating a sample in a refrigeration system according to any one of claims 17 to 19, comprising:
    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 heating the sample by using an external heat source, and determining that the average temperature of the sample is at a first temperature by using a temperature measurement unit; wherein the first temperature is greater than the second temperature.
  30. The method as recited in claim 29, further comprising:
    and detecting and adjusting the electrical parameter to enable the average temperature of the local temperature control area to reach a second temperature.
  31. The method of claim 29, wherein the step of providing the first information comprises,
    changing the second temperature to the first temperature over a predetermined period of time.
  32. The method of claim 29, wherein the predetermined period of time is within 10 ms.
  33. The method of claim 29, wherein the step of providing the first information comprises,
    the first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly converted from the liquid state to the amorphous solid state in the same environment and the amorphous solid state is continuously maintained.
  34. The method of claim 33, wherein the step of determining the position of the probe is performed,
    the first temperature is 0 ℃ to 40 ℃ and the second temperature is less than-140 ℃.
  35. A method for manipulating a sample using the sample testing system of claim 20, 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 placing layer;
    and detecting and adjusting the electrical parameter to a first preset 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 in the lowest temperature range which can be provided by the cryogenic cold source.
  36. The method as recited in claim 35, further comprising:
    adjusting an electrical parameter of a temperature control unit to a second predetermined range to heat the sample or to heat 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.
  37. The method according to claim 35 or 36, further comprising:
    and after the step of adjusting the electrical parameters of the temperature control unit to maintain the average temperature of the sample at a first temperature and the step of 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 the starting moment of adjusting the electrical parameters to a first preset range, and detecting and adjusting the electrical parameters to the first preset range at the starting moment so as to maintain the average temperature of the sample at a second temperature.
  38. The method as recited in claim 37, further comprising:
    after manipulating the sample, the sample is replaced.
  39. The method of claim 35, wherein the step of determining the position of the probe is performed,
    The first temperature is changed to the second temperature for a first predetermined period of time.
  40. The method of claim 35, wherein the electrical parameters of the temperature control unit are adjusted by an electronic device.
  41. The method of claim 35, wherein the step of determining the position of the probe is performed,
    changing the second temperature to the third temperature for a second predetermined period of time.
  42. The method of claim 41, wherein the second predetermined period of time is within 10 ms.
  43. The method of any one of claims 35-42, wherein,
    the first temperature is the liquid temperature of the sample, and the second temperature is the temperature at which the same sample is directly converted from the liquid state to the amorphous solid state in the same environment and the amorphous solid state is continuously maintained.
  44. The method of claim 43, wherein,
    the first temperature is 0 ℃ to 40 ℃ and the second temperature is less than-140 ℃.
  45. The method of claim 35, wherein the method is suitable for microscopic observation of a sample.
CN202180086244.4A 2020-12-28 2021-05-10 Freezing chip, freezing system, sample testing system and method Pending CN116685838A (en)

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