Supporting device for supporting a sample when freezing, freezing device and method

Abstract

The embodiment of the present disclosure discloses a support device for supporting a sample during freezing, a freezing device and a method, the support device comprising: a support layer in contact with a cold source; an interface layer on the support layer for placing a sample; a heat source with adjustable electrical parameters cooperating with the cold source for freezing the sample; wherein the binding force between the frozen sample and the interface layer or the binding force between the interface layer and the support layer is less than the maximum external force that the frozen sample can withstand. The above technical solution sets the interface layer on the support layer in contact with the cold source, the sample directly contacts the interface layer, realizes the separation of the frozen sample, is compatible with different types of carriers, the carrier cost is low, and the overall cost is reduced. Moreover, after the frozen sample is separated, subsequent observation and sample processing will be easier, and the operation is simplified.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedical technology, and in particular, to a support device for supporting a sample during freezing, a freezing device and a freezing method. BACKGROUND

[0002] Rapid freezing and heating technologies of biological samples have many important applications in the field of biomedicine, such as cell cryopreservation and revival, protein cryofixation characterization, etc.

[0003] Current biological freezing technologies mainly include plunge freezing, jetting freezing and high pressure freezing. Plunge freezing is the most commonly used sample preparation method in the industry. Plunge freezing usually fixes a sample stage (micro-grid) loaded with a biological sample at the front end of a sample rod, and quickly inserts the sample into a low-temperature liquid, such as liquid ethane or liquid nitrogen, by mechanical control, thereby completing the freezing of the biological sample. Jetting freezing usually transports a sample stage loaded with a biological sample to a specific position in a freezing chamber through a sample rod, and then uses high-pressure liquid nitrogen vapor to jet the sample at high speed, thereby completing the freezing of the biological sample. High pressure freezing is similar in principle to plunge freezing, which uses a low-temperature liquid to freeze the sample, but at the same time, a high pressure of about 2000 atmospheres is applied in the sample chamber to reduce the freezing temperature of water and inhibit the volume expansion during ice crystallization, thereby avoiding the damage of ice crystallization to the structure of the biological sample and preparing high-quality frozen biological samples.

[0004] However, plunge freezing has the following defects: since the entire sample needs to be inserted into a low-temperature liquid, it is not possible to selectively freeze specific areas of the sample during the freezing process, and real-time microscopic observation cannot be performed in situ during the freezing process. Jetting freezing improves the heat transfer efficiency by using high-speed jetting of low-temperature liquid to cool. High pressure freezing is similar in principle to the above two freezing methods, and the freezing effect is better and the sample quality is higher due to the inhibition of ice crystallization by high pressure. However, jetting freezing and high pressure freezing also have the defects of not being able to perform real-time microscopic observation and local selective freezing. These defects limit further in-depth research on frozen biological samples. SUMMARY

[0005] To solve the problems in the related art, the embodiments of the present disclosure provide a support device for supporting a sample during freezing, a freezing device and a freezing method.

[0006] In a first aspect, the embodiments of the present disclosure provide a support device for supporting a sample during freezing.

[0007] Specifically, the support device for supporting a sample during freezing comprises:

[0008] a support layer in contact with the cold source;

[0009] an interface layer on the support layer for placing a sample;

[0010] a heat source with adjustable electrical parameters, cooperating with the cold source, for freezing the sample;

[0011] wherein the binding force between the frozen sample and the interface layer or the binding force between the interface layer and the support layer is less than the maximum external force that the frozen sample can withstand.

[0012] Optionally, the heat source is an external heat source or the support layer is provided with a heat source.

[0013] Optionally, when the heat source is an external heat source, the support layer is a freezing substrate or the support layer is a composite layer composed of a freezing substrate and a thermal resistance layer on the freezing substrate.

[0014] Optionally, the support layer comprises a heating layer arranged below the interface layer; the heating layer is the heat source.

[0015] Optionally, the support layer further comprises:

[0016] a freezing substrate provided with a thermal resistance layer;

[0017] wherein the heating layer is on the thermal resistance layer.

[0018] Optionally, the interface layer is directly laid on the support layer; or the surface of the support layer is subjected to hydrophobic treatment to form the interface layer.

[0019] Optionally, the material of the interface layer is isopentane, n-hexane, a hydrophobic material, or the interface layer is a hydrophobic structure.

[0020] Optionally, the material of the heating layer is metal, semiconductor material or conductive compound.

[0021] In a second aspect, the embodiments of the present disclosure provide a freezing device.

[0022] Specifically, the freezing device comprises:

[0023] a cold source for freezing a sample;

[0024] a support layer in contact with the cold source;

[0025] an interface layer on the support layer;

[0026] The binding force between the frozen sample and the interface layer or the binding force between the interface layer and the support layer is less than the maximum external force that the frozen sample can withstand.

[0027] Optionally, the device further comprises:

[0028] The heat source, which can be adjusted in electrical parameters, cooperates with the cold source to freeze the sample.

[0029] The heat source is an external heat source or part of the support layer is configured as a heat source.

[0030] Optionally, when the heat source is an external heat source, the support layer is a freezing substrate or the support layer is a composite layer composed of a freezing substrate and a thermal resistance layer located on the freezing substrate.

[0031] Optionally, the support layer comprises a heating layer arranged below the interface layer; the heating layer is the heat source.

[0032] Optionally, the support layer further comprises:

[0033] A freezing substrate, on which a thermal resistance layer is arranged;

[0034] The heating layer is located on the thermal resistance layer.

[0035] Optionally, the device further comprises:

[0036] A sample carrier located on one side of the interface layer for placing a sample;

[0037] The distance between the sample carrier and the interface layer can be adjusted.

[0038] Optionally, the device further comprises:

[0039] At least a first support structure located between the sample carrier and the interface layer.

[0040] Optionally, the heights of the first support structures are the same or different, and the shapes of the first support structures are the same or different.

[0041] Optionally, at least one second support structure is arranged on the sample carrier and / or the interface layer.

[0042] Optionally, the heights of the second support structures are the same or different, and the shapes of the second support structures are the same or different.

[0043] Optionally, the device further comprises:

[0044] A driving unit for driving the sample to move in the space formed between the interface layer and the sample carrier.

[0045] Optionally, the driving unit employs an EWOD device to drive the sample to move.

[0046] Optionally, the interface layer is directly laid on the support layer; or the interface layer is laid on the surface of the support layer after the surface of the support layer is treated by hydrophobic treatment.

[0047] Optionally, the material of the interface layer is isopentane, n-hexane, or a hydrophobic material, or the interface layer is a hydrophobic structure.

[0048] Optionally, the material of the heating layer is a metal, a semiconductor material, or a conductive compound.

[0049] Optionally, the sample carrier is a mesh coated with a film structure.

[0050] Optionally, the sample is an aqueous sample.

[0051] In a third aspect, the embodiments of the present disclosure provide a freezing system using the freezing device of any one of the second aspect.

[0052] Specifically, the freezing system comprises an objective lens for observing a normal-temperature sample state, a freezing process, and a frozen sample through the freezing substrate or the sample carrier.

[0053] Optionally, the objective lens is an oil immersion objective lens.

[0054] Optionally, the oil immersion objective lens is in contact with the freezing substrate; or the oil immersion objective lens is in direct contact with the sample carrier.

[0055] In a fourth aspect, the embodiments of the present disclosure provide a method for operating a sample using the freezing system of the third aspect.

[0056] Specifically, the method comprises the following steps:

[0057] a step of freezing the sample;

[0058] a step of separating the frozen sample from the freezing device by using an external force.

[0059] Optionally, before the step of freezing the sample, the method further comprises:

[0060] a step of moving the sample carrier to control the thickness of the sample.

[0061] Optionally, the step of freezing the sample comprises:

[0062] adjusting an electrical parameter of the heat source to maintain the average temperature of the sample stable at a first temperature;

[0063] The electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the cold source can provide.

[0064] Optionally, before detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, the method further comprises:

[0065] The sample is treated by increasing the temperature from the first temperature to the third temperature.

[0066] Optionally, the first temperature is the liquidus temperature of the sample, and the second temperature is the temperature at which the same sample is directly transformed from liquid to glassy state under the same environment and continuously maintains the glassy state.

[0067] Optionally, the first temperature is from 0°C to 40°C, and the second temperature is lower than the critical temperature at which the sample forms a glassy state.

[0068] Optionally, it also includes:

[0069] Steps for observing frozen samples.

[0070] Optionally, it also includes:

[0071] The step of thawing the frozen sample by heating it with a heat source.

[0072] In a fifth aspect, an embodiment of the present disclosure provides a method for operating a sample using the freezing system described in the third aspect.

[0073] Specifically, the method comprises the following steps:

[0074] placing the sample in the channel formed between the interface layer and the sample carrier;

[0075] Steps for freezing samples;

[0076] The step of separating the frozen sample from the freezing device by using external force.

[0077] Optionally, the step of freezing the sample comprises:

[0078] adjusting electrical parameters of the heat source to maintain the average temperature of the sample at a first temperature;

[0079] The electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the cold source can provide.

[0080] Optionally, the detecting and adjusting the electrical parameter to the first predetermined range to adjust the average temperature of the sample to be before the second temperature, the method further comprises:

[0081] Raising the sample from the first temperature to a third temperature for processing.

[0082] Optionally, the first temperature is a liquid temperature of the sample, and the second temperature is a temperature that makes the same sample directly change from a liquid state to a glass state under the same environment and continuously maintain the glass state.

[0083] Optionally, the first temperature is 0-40℃, and the second temperature is lower than a critical temperature at which the sample forms a glass state.

[0084] Optionally, the method further comprises:

[0085] The step of observing the frozen sample.

[0086] Optionally, the method further comprises:

[0087] The step of thawing the frozen sample by heating the frozen sample with a heat source.

[0088] The technical scheme provided by the embodiments of the present disclosure can include the following beneficial effects:

[0089] (1) The support device of the embodiments of the present disclosure, by setting an interface layer on the support layer in contact with the cold source, the sample directly contacts the interface layer, and the appropriate interface layer material is selected, so that the frozen sample can be separated from the interface layer without being damaged, or part of the interface layer is transferred together, realizing the separation of the frozen sample, and being compatible with different types of carriers, the cost of the carrier is low, and the overall cost is reduced. Moreover, after the frozen sample is separated, subsequent observation and sample processing will be easier, and the operation is simplified.

[0090] (2) The freezing device of the embodiments of the present disclosure, the interface layer is preferably a liquid material, which can reduce the flatness requirement of the surface of the support layer, such as the heating layer or the thermal resistance layer, thereby further improving the sample surface on the basis of the existing flatness, so that the surface of the frozen sample is more flat, which is beneficial to subsequent optical and electronic imaging, especially for high-resolution imaging.

[0091] (3) The freezing system of the embodiments of the present disclosure, by setting the first support structure and the second support structure to adjust the thickness of the frozen sample, thereby being beneficial to improve the freezing speed of the sample and facilitate subsequent observation and sample processing.

[0092] (4) The method for operating a sample of the freezing system of the embodiments of the present disclosure can separate the frozen sample from the interface layer after the sample is frozen, and the subsequent observation and sample processing will be easier, and the operation is simplified. In addition, the frozen sample can be observed in cooperation with an oil immersion objective.

[0093] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0094] Other features, objects, and advantages of the present disclosure will become more apparent from the following detailed description of the non-limiting embodiments in conjunction with the accompanying drawings. In the drawings:

[0095] Figure 1 A schematic diagram showing sample freezing and observation of the present disclosure is shown;

[0096] Figure 2 A support device for supporting a sample during freezing of the present disclosure is shown;

[0097] Figures 3a-3c A schematic diagram showing the principle of transferring a frozen sample is shown;

[0098] Figure 4 A schematic diagram showing the structure of a freezing device of the present disclosure is shown;

[0099] Figure 5 A schematic diagram showing the structure of another freezing device of the present disclosure is shown;

[0100] Figure 6 A flowchart showing the separation of a frozen sample from an interface layer is shown;

[0101] Figures 7a-7d A schematic diagram showing the principle of controlling the thickness of a frozen sample by a first support structure and a second support structure is shown;

[0102] Figure 8 A schematic diagram showing the structure of a freezing system of the present disclosure is shown;

[0103] Figures 9a-9b A schematic diagram showing an oil immersion objective observing a frozen sample from the sample side is shown;

[0104] Figure 10 A flowchart showing a method for operating a sample using a freezing system is shown;

[0105] Figure 11 A flowchart showing another method for operating a sample using a freezing system is shown. DETAILED DESCRIPTION

[0106] Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so as to be easily carried out by one of ordinary skill in the art. Also, portions irrelevant to the description of the exemplary embodiments are omitted in the accompanying drawings for the sake of clarity.

[0107] In the present disclosure, it should be understood that terms such as "include" or "have" are intended to indicate that there are features, numbers, steps, actions, parts, or combinations thereof disclosed in the specification, and do not exclude the possibility that one or more other features, numbers, steps, actions, parts, or combinations thereof exist or are added.

[0108] It is further noted that the embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0109] The inventors of the present disclosure propose a sample freezing method based on a freezing chip, which is different from the three ways of plug-in freezing, spray freezing, and high-pressure freezing in the principle of freezing samples. As shown in Figure 1 The difference is that the heating element built-in the freezing chip has a heating function, the sample is placed on the freezing chip, the cold source provides a low-temperature environment for the freezing chip, the sample is kept at a higher temperature by the heating element of the chip (to prevent the sample from being frozen), the sample is placed under the observation equipment, when it is needed to observe a certain form of the sample, the electrical parameters of the heating element are adjusted, the heat of the sample is quickly transferred to the cold source to achieve rapid freezing of the sample. The frozen sample continues to be observed under the observation equipment.

[0110] In the process of freezing the sample and observing it, the sample freezing and observation are simultaneously realized on the freezing chip, that is, the freezing chip integrates the functions of sample freezing and sample carrier. The inventors found that, since the chip is a consumable, it needs complex processes for placing the heating element and arranging the electrical connection structure, resulting in high cost. In order to prolong the service life of the chip and reduce the cost, the present disclosure proposes a support device for supporting a sample during freezing, a freezing device, a freezing system, and a method for operating a sample based on the freezing system. The inventive concept of the present disclosure is to separate the functions of sample freezing and sample carrier of the chip, after the sample is frozen, it can be separated from the contact surface of the chip and transferred to the sample carrier, and the subsequent observation is carried out on the sample carrier, thereby achieving the purpose of the present disclosure.

[0111] The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0112] Figure 2 The support device for supporting a sample during freezing of the present disclosure is shown. Figures 3a-3c The principle diagram showing the transfer of the frozen sample is shown.

[0113] As Figure 2As shown, the support device 10 for supporting the sample during freezing comprises a support layer 11 and an interface layer 12. The support layer 11 comprises a freezing substrate 111, a thermal resistance layer 112 and a heating layer 113. The thermal resistance layer 112 and the heating layer 113 are sequentially arranged on the freezing substrate 111. The interface layer 12 is arranged on the heating layer 113. In the present disclosure, the freezing substrate 111 serves as the mechanical carrier part of the support device 10, and the material used can be silicon, silicon carbide, metal, diamond or ceramic, etc. The thermal resistance layer 112 is arranged to provide a temperature gradient, and the material used can be silicon oxide, silicon nitride, polymer, etc. The material of the heating layer 113 is usually an electrically conductive material, such as metal (aluminum, copper, platinum, etc.), an electrically conductive compound (titanium nitride, indium tin oxide, etc.) or a semiconductor material (silicon, silicon carbide, etc.). The support layer 11 can be realized by chip manufacturing process, and specific details can be referred to the prior art, which will not be described herein.

[0114] Please refer to Figures 3a to 3c , the support layer 11 is in contact with a cold source, which can be a low-temperature liquid such as liquid nitrogen, can be a functional unit with a refrigeration structure, or can be a high-performance heat conduction structure. The sample is placed on the sample carrier 13 and supported by the interface layer 12. The sample carrier 13 can be a mesh coated with a film structure or a chip with a suspended film structure. The heating layer 113 serves as a heat source to cooperate with the cold source to realize rapid freezing of the sample. The principle is as described above. The heat source is used to maintain the temperature of the surface of the interface layer 12 sufficient to prevent the sample from being frozen. When the sample needs to be frozen, the parameters of the heat source can be adjusted to transfer heat to the cold source to realize rapid freezing of the sample. After the sample is frozen, the sample carrier 13 is transferred, for example, the sample is separated from the interface layer 12 (as shown in Figure 3b ), or part of the interface layer 12 is carried away (as shown in Figure 3c ), or even the entire interface layer 12 is carried away from the support layer 11, and then transferred to the observation equipment through the sample carrier 13 for observation.

[0115] According to an embodiment of the present disclosure, the heat source can be the heating layer 113 in the support layer 11, or an external heat source. The external heat source can use radiation heating. The external heat source can directly heat the sample itself, or can heat the support layer 11 to indirectly heat the sample on the interface layer 12. The heating mode of the heating layer 113 and the external heat source can be selected as needed. When the heating layer 113 is used, the thermal resistance layer 112 between the heating layer 113 and the freezing substrate 111 can be omitted in some cases. Similarly, when the external heat source is used, the support layer 11 can be the freezing substrate 111, or a composite layer composed of the freezing substrate 111 and the thermal resistance layer 112, which is not limited in the present disclosure.

[0116] The support device provided by the present disclosure sets an interface layer between the sample and the support layer, which aims to separate the support layer after the sample is frozen. If the sample is directly frozen on the support layer, when the frozen sample is too tightly combined with the support layer, the force applied to the sample carrier may exceed the maximum external force that the frozen sample can withstand, which easily damages the integrity of the frozen sample and is not conducive to subsequent observation.

[0117] By selecting a suitable interface material as the interface layer, within the range of the maximum external force that the sample can withstand (to avoid sample damage), when the external force applied to the frozen sample is greater than the binding force between the frozen sample and the interface layer, the frozen sample can be directly separated from the interface layer or part of the interface layer is transferred together. In some cases, if the external force applied to the frozen sample is greater than the binding force between the interface layer and the support layer, part or all of the interface layer can also be transferred together to achieve the separation of the frozen sample.

[0118] According to the embodiments of the present disclosure, an interface layer 12 can be directly laid on the support layer 11, or the surface of the support layer 11 can be treated to form an interface layer 12.

[0119] The material of the interface layer can be:

[0120] 1) single-layer material or composite material;

[0121] 2) material that is liquid at room temperature, liquid or solid at sample freezing temperature;

[0122] 3) material that is solid at room temperature, solid at sample freezing temperature.

[0123] The room temperature refers to room temperature, for example, 20-30℃, and the sample freezing temperature can be, for example, the critical temperature at which the liquid forms a glass state and the temperature below it, such as -140℃.

[0124] The interface layer in the present disclosure is preferably a material with thin thickness, high thermal conductivity, and small thermal melting, so as to improve the freezing speed. The interface layer is also preferably a liquid material. By setting the interface layer, the sample is placed on the interface layer, which can also reduce the requirement for the flatness of the surface of the support layer, such as the heating layer or the thermal resistance layer, thereby further improving the sample surface on the basis of the existing flatness, so that the sample surface after freezing is more flat, which is conducive to subsequent optical and electronic imaging, especially for high-resolution imaging.

[0125] For different materials of the interface layer and the support layer, direct laying (hereinafter referred to as coating process) or surface treatment process can be used to form the interface layer on the support layer. The following provides several exemplary descriptions, which can be understood as not constituting a limitation on the present disclosure.

[0126] The first mode: the interface layer is isopentane, which is in liquid state at room temperature and in liquid state at the sample freezing temperature, and the film is formed on the support layer. The thickness of the film is preferably less than 1 um. The use of a thin interface layer can ensure that the sample freezing speed is not affected.

[0127] The second mode: the interface layer is n-hexane, which is in liquid state at room temperature and in solid state at the sample freezing temperature, and the film is formed on the support layer. The thickness of the film is referred to isopentane.

[0128] The third mode: the interface layer is a hydrophobic material, such as Teflon, which is formed on the support layer. The thickness is preferably 100 nm. It should be noted that when selecting the hydrophobic material, its mechanical reliability at low temperature and whether the hydrophobic contact angle is large enough need to be considered, especially the performance stability over a long period of use. Teflon materials have better low-temperature mechanical reliability and larger hydrophobic angle, and are suitable for application. Polystyrene materials have poor performance in the above two aspects, so the hydrophobic material in the present disclosure does not include polystyrene materials.

[0129] The fourth mode: the interface layer is a hydrophobic material molecule, such as fluorosilane, which is formed on the support layer. The thickness is a monolayer.

[0130] The fifth mode: the interface layer is a hydrophobic structure, such as a nano-array structure with super-hydrophobic properties generated after surface treatment of the support layer. The thickness is preferably 100 nm.

[0131] Based on the same or similar design idea, the present disclosure also provides a freezing device. The specific technical details can refer to the embodiments shown in Figure 2 、 Figures 3a-3c , which will not be described here.

[0132] Figure 4 The structure of the freezing device of the present disclosure is shown in the schematic diagram.

[0133] As Figure 4 shown, the freezing device 20 includes a cold source 21, a support layer 22, and an interface layer 23. The cold source 21, such as liquid nitrogen, provides a low temperature below -190℃ for freezing the sample. The support layer 22 is in contact with the cold source 21, and the interface layer 23 is on the support layer 22. The binding force between the frozen sample and the interface layer 23 or the binding force between the interface layer 23 and the support layer 22 is less than the maximum external force that the frozen sample can withstand, so that the frozen sample and the interface layer 23 are separated. In the present disclosure, the sample can be an aqueous sample.

[0134] According to an embodiment of the present disclosure, the freezing device further comprises a heat source with adjustable electrical parameters, which cooperates with the cold source to freeze the sample; the heat source is an external heat source or a part of the support layer.

[0135] According to an embodiment of the present disclosure, when the heat source is an external heat source, the support layer is a freezing substrate or a composite layer composed of a freezing substrate and a thermal resistance layer on the freezing substrate.

[0136] According to an embodiment of the present disclosure, the support layer comprises a heating layer arranged below the interface layer; the heating layer is the heat source.

[0137] According to an embodiment of the present disclosure, the support layer further comprises a freezing substrate with a thermal resistance layer arranged thereon; wherein the heating layer is arranged on the thermal resistance layer.

[0138] According to an embodiment of the present disclosure, the interface layer is directly arranged on the support layer; or the interface layer is arranged on the surface of the support layer after the surface of the support layer is subjected to hydrophobic treatment.

[0139] According to an embodiment of the present disclosure, the material of the interface layer is isopentane, n-hexane or a hydrophobic material, or the interface layer is a hydrophobic structure.

[0140] According to an embodiment of the present disclosure, the material of the heating layer is a metal, a semiconductor material or an electrically conductive compound.

[0141] Figure 5 A structural schematic diagram of another freezing device of the present disclosure is shown.

[0142] As shown in Figure 5 , the freezing device 30 comprises a cold source 31, a support layer 32, an interface layer 33 and a sample carrier 34. The cold source 31, the support layer 32 and the interface layer 33 are arranged separately from the sample carrier 34 as a whole, and the sample carrier 34 is located on one side of the interface layer 33. The cold source 31, such as liquid nitrogen, provides a low temperature lower than the critical temperature at which a liquid forms a glass state, for freezing the sample. The support layer 32 is in contact with the cold source 31, and the interface layer 33 is arranged on the support layer 32. The sample can be placed on the sample carrier 34 or on the interface layer 33. After the sample is frozen, the sample carrier 34 is used to separate the sample from the interface layer 33.

[0143] Figure 6 A flowchart showing the separation of the frozen sample from the interface layer is shown.

[0144] As shown in Figure 6 , the process of freezing the sample and separating the sample from the interface layer is as follows:

[0145] Sample placement: place sample A on the sample carrier 34, and the sample is in a liquid state at this time (the sample is in a water environment).

[0146] Sample contact interface layer and extrusion thinning: the sample is in contact with and between the sample carrier 34 and the interface layer 33, as the sample carrier 34 approaches the interface layer 33 in a direction perpendicular to the surface, the thickness of the sample is extruded and thinned. The thickness of the sample is controlled by the distance between the sample carrier 34 and the interface layer 33, and the final thickness is controlled between tens of nanometers and hundreds of microns, depending on the application requirements of the sample, the thermal properties of the sample, or the freezing phase change performance, which is not limited here. The temperature of the sample and its contact interface layer 33 throughout the process is higher than the solidification temperature of the sample (the temperature at which water freezes), so that the sample remains liquid.

[0147] Sample freezing: adjust the electrical parameters of the heat source (heating layer 113), for example, by reducing the heating power of the heat source, so that the temperature of the interface layer 33 and the sample decreases to the temperature of the cold source until a new temperature balance is reached. The new sample temperature can be equal to or higher than the cold source temperature, and the cooling rate is affected by power adjustment, thermal properties of the cold source (and materials above it), and the surrounding environment (such as air temperature). The thermal properties of the cold source (and materials above it) determine the upper limit of the cooling rate. Through testing, the cooling rate can reach the order of 1E5℃ / s.

[0148] Freezing sample separation: the frozen sample is transferred by the sample carrier 34.

[0149] According to embodiments of the present disclosure, the distance between the sample carrier 34 and the interface layer 33 can be adjusted to control the thickness of the sample. The control of the sample thickness has a great influence on the freezing quality, under the same conditions, the greater the thickness, the slower the freezing speed, and the poorer the freezing quality; but a larger sample thickness can accommodate a higher sample. Therefore, the thickness of the sample needs to be adjustable or limited by a pre-set structure. There are several different ways to control the thickness of the sample, one way is to adjust the relative distance between the sample carrier 34 and the interface layer 33 by high-precision sliding table, piezoelectric element or gas pressure control device, etc., and to extrude the sample by external force. One way is to use the surface tension of the sample to spontaneously thin. One way can be to provide a first support structure 35 between the sample carrier 34 and the interface layer 33, the heights of the plurality of first support structures 35 can be the same or different, and the shapes of the plurality of first support structures 35 can be the same or different, and the different heights and shapes of the first support structures 35 are used to control the morphology of the frozen sample. Another way can be to provide at least one second support structure 36 on the sample carrier 34 and / or the interface layer 33, the heights of the plurality of second support structures 36 can be the same or different, and the shapes of the plurality of second support structures 36 can be the same or different.

[0150] Figures 7a-7d The principle of the first support structure and the second support structure controlling the thickness of the frozen sample is shown. As shown in FIG. 6, the first support structure 35 is provided between the sample carrier 34 and the interface layer 33, and the second support structure 36 is provided on the interface layer 33. Figure 7aAs shown, the first support structure 35 is located between the sample carrier 34 and the interface layer 33, and the thickness of the sample A is controlled by the first support structure 35. When the sample carrier 34 is separated from the interface layer 33, the first support structure 35 is transferred into the frozen sample. Figure 7b 、 Figure 7c Then, the second support structure 36 is arranged on the sample carrier 34 and the interface layer 33 respectively, and the thickness of the sample A is controlled by the second support structure 36. Figure 7d The second support structure 36 with different heights is shown. When the sample carrier 34 is separated from the interface layer 33, the frozen sample presents different heights.

[0151] According to an embodiment of the present disclosure, the freezing device 30 further comprises a driving unit for driving the sample to move in the space formed between the interface layer 33 and the sample carrier 34. Specifically, the driving unit drives the sample to move by using an EWOD (electrowetting-on-dielectric) device or by using an external force.

[0152] Based on the same or similar design idea, the present disclosure further provides a freezing system comprising any one of the above freezing devices. The specific technical details of the freezing device can be referred to the above embodiments, and will not be described here. Figure 4 、 Figure 5 The above embodiments are not described here.

[0153] The freezing system further comprises an objective lens. The objective lens is used to observe the normal-temperature sample state, the freezing process and the frozen sample through the freezing substrate or the sample carrier. Further, the objective lens can be an oil immersion objective lens. The oil immersion objective lens is usually used in the case of high-resolution optical observation. When used, the oil immersion objective lens and the observed object are in contact through a liquid (usually oil), so that heat exchange exists between them.

[0154] Figure 8 、 Figure 9a 、 Figure 9b The freezing system 40 shown in FIG. 8 is taken as an example to illustrate the embodiment in which the freezing device cooperates with the oil immersion objective lens 41 to perform sample observation. The support layer 32 of the freezing system 40 is composed of the freezing substrate 111, the thermal resistance layer 112 and the heating layer 113.

[0155] Figure 8 The frozen sample is observed from the side of the freezing substrate. Figure 9a 、 Figure 9b The frozen sample A is observed from the side of the sample carrier. In the figure, B is a carrier for fixing the sample carrier. Figure 8As shown, the oil immersion objective 41 is in contact with the frozen substrate 111 after being immersed in the oil 42 for sample observation, which is suitable for low-temperature objective lenses that can work at the temperature of the cold source. At this time, the frozen substrate 111 is in contact with the cold source through the laterally extending part that does not contact the lens, which is suitable for observing the normal state and freezing process of the sample. As shown in Figure 9a 、 9b As shown, the oil immersion objective 41 is placed on one side of the sample carrier 34, which can be used to observe the sample before the freezing starts. After that, the oil immersion objective 41 is moved away from the carrier for a short time before the freezing starts, and then the sample is frozen, which avoids the temperature of the objective lens from falling out of the usable range, and at the same time avoids the heat capacity of the objective lens and the objective oil affecting the cooling speed of the sample.

[0156] Based on the same or similar design idea, the disclosure also provides a method for operating a sample by using the above-mentioned freezing system. The specific technical details of the freezing system can be referred to the above-mentioned embodiments, which will not be repeated here.

[0157] As shown in Figure 10 The method for operating a sample by using the above-mentioned freezing system comprises the following steps:

[0158] S110: a step of freezing the sample;

[0159] S120: a step of separating the frozen sample from the freezing device by using an external force.

[0160] According to an embodiment of the disclosure, before the step S110 of freezing the sample, the method further comprises:

[0161] The step of moving the sample carrier controls the thickness of the sample.

[0162] According to an embodiment of the disclosure, the step S110 of freezing the sample comprises:

[0163] Adjusting the electrical parameters of the heat source to maintain the average temperature of the sample at a first temperature;

[0164] Detecting and adjusting the electrical parameters to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that can be provided by the cold source.

[0165] In the present disclosure, the controller is used to control the heat source to automatically increase the current I heater The resistance heating is performed to maintain the average temperature of the sample at a first temperature (such as 30℃), and the typical current value range is I heater = 50-100 mA, R heater The typical power (R heater *I heater 2) about 0.3 W; when freezing is needed, the controller sends a signal to suddenly reduce the current Iheater to 0.1-1.0 mA, the sample temperature will rapidly decrease, R heater also sharply decreases to about 1 / 7 of the room temperature, R heater During the whole cooling process, the control circuit maintains a small constant current (0.1-1.0 mA) for continuously measuring the change of R heater , as a reference of the temperature change over time. After the freezing is completed, the control circuit maintains a small current (0.1-1.0 mA) to keep the average temperature of the sample at the second temperature (such as the temperature of the normal pressure liquid nitrogen) and continuously monitors the change of R heater as a reference of the sample temperature.

[0166] In the present disclosure, the electrical parameter is detected and adjusted to the first predetermined range to adjust the average temperature of the sample to the second temperature, and the method further comprises: raising the sample from the first temperature to a third temperature for processing.

[0167] For example, one application scenario is to control the speed of chemical reactions or life activities by using the activity of biological proteins or other macromolecular materials at different temperatures. Specifically, by using the temperature activity of cell membrane proteins, the cell sample is contacted with the virus sample at the first temperature (such as 4°C), at which time the virus is attached to the cell membrane and combined with specific membrane proteins, but the virus cannot invade the cell due to the low activity of the proteins. Then, by rapidly raising the sample temperature to a third temperature such as 37°C, the protein activity is activated, and the virus begins to invade the cell. After a short delay time (usually a few seconds), the sample is rapidly cooled to the second temperature to freeze the instantaneous state of the virus invading the cell. Then, observation can be performed for studying the invasion mechanism of the virus.

[0168] According to an embodiment of the present disclosure, the first temperature is the liquid temperature of the sample, and the second temperature is the temperature that makes the same sample directly change from the liquid state to the glass state in the same environment and continuously maintain the glass state.

[0169] According to an embodiment of the present disclosure, the first temperature is 0-40°C, and the second temperature is lower than the critical temperature at which the sample forms the glass state, for example, lower than -140°C.

[0170] According to an embodiment of the present disclosure, the method further comprises:

[0171] S130: a step of observing the frozen sample.

[0172] In the present disclosure, the frozen sample can be observed by using a general objective lens, and the general objective lens is spaced apart from the frozen sample for observation. In order to improve the resolution, an oil immersion objective lens can also be used for observation, and the observation method of the oil immersion objective lens can refer to Figure 8 ,Figure 9a 、 Figure 9b In this way, further description is omitted here.

[0173] According to an embodiment of the present disclosure, the method further comprises:

[0174] S140: heating the frozen sample by using the heat source to realize thawing.

[0175] Based on the same or similar design idea, the present disclosure further provides a method for operating a sample by using the above-mentioned freezing system. The specific technical details of the freezing system can refer to the above-mentioned embodiments, and further description is omitted here.

[0176] As shown in Figure 11 , the method for operating a sample by using the freezing system comprises the following steps:

[0177] S210: placing the sample in the channel formed between the interface layer and the sample carrier;

[0178] S220: freezing the sample;

[0179] S230: separating the frozen sample from the freezing device by using an external force.

[0180] According to an embodiment of the present disclosure, the step S220 of freezing the sample comprises:

[0181] adjusting the electrical parameters of the heat source to maintain the average temperature of the sample at a first temperature;

[0182] detecting and adjusting the electrical parameters to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the cold source can provide.

[0183] In the present disclosure, the controller is used to control the heat source to automatically increase the current I heater to perform resistance heating, and maintain the average temperature of the sample at a first temperature (such as 30℃), at which the typical current value range is I heater = 50-100 mA, R heater The typical power (R heater *I heater 2 ) is about 0.3 W; when freezing is required, the controller sends a signal to suddenly reduce the current I heater to 0.1-1.0 mA, and the sample temperature will rapidly decrease, and R heater will also sharply decrease to about 1 / 7 of R heater at room temperature, and during the whole cooling process, the control circuit maintains a small constant current (0.1-1.0 mA) for continuous measurement of R heatertemperature change, as a reference of temperature change over time. After the freezing is completed, the control circuit maintains a small current (0.1-1.0 mA) to maintain the average temperature of the sample at the second temperature (such as the temperature of the normal pressure liquid nitrogen) and continuously monitors the change of R heater temperature change, as a reference of temperature change over time. After the freezing is completed, the control circuit maintains a small current (0.1-1.0 mA) to maintain the average temperature of the sample at the second temperature (such as the temperature of the normal pressure liquid nitrogen) and continuously monitors the change of R

[0184] In the present disclosure, the electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to the second temperature, and the method further comprises: raising the sample from the first temperature to a third temperature for processing.

[0185] For example, one application scenario is to control the speed of chemical reactions or life activities by using the activity of biological proteins or other macromolecular materials at different temperatures. Specifically, by using the temperature activity of cell membrane proteins, the cell sample is contacted with the virus sample at a first temperature (such as 4°C), at which time the virus is attached to the cell membrane and combined with specific membrane proteins, but the virus cannot invade the cell due to the low activity of the proteins. Then, by rapidly raising the temperature of the sample to a third temperature such as 37°C, the activity of the proteins is activated, and the virus begins to invade the cell. After a short delay time (usually a few seconds), the sample is rapidly cooled to the second temperature to freeze the instantaneous state of the virus invading the cell. Then, observation can be performed for studying the invasion mechanism of the virus.

[0186] According to an embodiment of the present disclosure, 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 a glass state in the same environment and continuously maintained in the glass state.

[0187] According to an embodiment of the present disclosure, the first temperature is 0-40°C, and the second temperature is lower than the critical temperature at which the liquid forms a glass state.

[0188] According to an embodiment of the present disclosure, the method further comprises:

[0189] S240: a step of observing the frozen sample.

[0190] In the present disclosure, a general objective lens can be used to observe the frozen sample, and the general objective lens is spaced apart from the frozen sample for observation. In order to improve the resolution, an oil immersion objective lens can also be used for observation, and the observation method of the oil immersion objective lens can refer to the method of the oil immersion objective lens in the prior art, which will not be described here in detail. Figure 8 、 Figure 9a 、 Figure 9b

[0191] According to an embodiment of the present disclosure, the method further comprises:

[0192] S250: a step of thawing the frozen sample by heating the frozen sample with a heat source.

[0193] ​The above description is merely that of the preferred embodiments of the present disclosure and a description of the technical principles of the present disclosure. It should be understood by those skilled in the art that the inventive scope involved in the present disclosure is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by the combinations of the above technical features or equivalent features without departing from the inventive concept. For example, the technical solutions formed by the mutual replacement of the above features and the technical features with similar functions disclosed in the present disclosure (but not limited to) without departing from the inventive concept.

Claims

1. A support device for supporting a sample during freezing, characterized in that: include: A support layer in contact with the cooling source; an interface layer, located on the support layer and used for placing a sample; A heat source with adjustable electrical parameters, cooperating with the cold source, for freezing the sample; Among them, the bonding force between the frozen sample and the interface layer or the bonding force between the interface layer and the support layer is less than the maximum external force that the frozen sample can withstand; the heat source is an external heat source or the support layer is provided with a heat source; the interface layer is directly laid on the support layer; or the interface layer is formed after the surface of the support layer is hydrophobically treated.

2. The support device according to claim 1, characterized in that When the heat source is an external heat source, the support layer is a frozen substrate or the support layer is a composite layer consisting of a frozen substrate and a thermal resistance layer located on the frozen substrate.

3. The supporting device according to claim 1, characterized in that The support layer includes: a heating layer arranged below the interface layer; the heating layer is the heat source.

4. The supporting device according to claim 3, characterized in that The support layer further comprises: a frozen substrate having a thermal resistance layer disposed thereon; Wherein, the heating layer is located on the thermal resistance layer.

5. The supporting device according to claim 1, characterized in that The material of the interface layer is isopentane, n-hexane, or a hydrophobic material, or the interface layer is a hydrophobic structure.

6. The supporting device according to claim 3, characterized in that The material of the heating layer is metal, semiconductor material or conductive compound.

7. A refrigeration device, characterized in that: include: Cold source, used to freeze samples; a support layer in contact with the heat sink; an interface layer located on the support layer; The bonding force between the frozen sample and the interface layer or the bonding force between the interface layer and the support layer is less than the maximum external force that the frozen sample can withstand; the interface layer is directly laid on the support layer; or the interface layer is laid after the surface of the support layer is hydrophobically treated; Also includes: A heat source with adjustable electrical parameters, cooperating with the cold source, for freezing the sample; The heat source is an external heat source or a portion of the support layer is configured as a heat source.

8. The refrigeration device according to claim 7, characterized in that When the heat source is an external heat source, the support layer is a frozen substrate or the support layer is a composite layer consisting of a frozen substrate and a thermal resistance layer located on the frozen substrate.

9. The refrigeration device according to claim 7, characterized in that The support layer includes: a heating layer arranged below the interface layer; the heating layer is the heat source.

10. The refrigeration device according to claim 9, characterized in that The support layer further comprises: a frozen substrate having a thermal resistance layer disposed thereon; Wherein, the heating layer is located on the thermal resistance layer.

11. The freezing device according to any one of claims 7 to 10, characterized in that: Also includes: A sample carrier, located on one side of the interface layer, for placing samples; Wherein, the distance between the sample carrier and the interface layer can be adjusted.

12. The refrigeration device according to claim 11, characterized in that Also includes: At least a first support structure is positioned between the sample carrier and the interface layer.

13. The refrigeration device according to claim 12, characterized in that The plurality of first supporting structures may have the same or different heights and the same or different shapes.

14. The refrigeration device according to claim 11, characterized in that At least one second supporting structure is provided on the sample carrier and / or the interface layer.

15. The refrigeration device according to claim 14, characterized in that The plurality of second support structures may have the same or different heights and the same or different shapes.

16. The refrigeration device according to claim 11, characterized in that Also includes: The driving unit is used to drive the sample to move in the space formed between the interface layer and the sample carrier.

17. The refrigeration device according to claim 16, characterized in that The driving unit uses an EWOD device to drive the sample to move.

18. The freezing device according to claim 7, characterized in that The material of the interface layer is isopentane, n-hexane, or a hydrophobic material, or the interface layer is a hydrophobic structure.

19. The refrigeration device according to claim 9, characterized in that The material of the heating layer is metal, semiconductor material or conductive compound.

20. The freezing device according to claim 7, characterized in that The sample is an aqueous phase sample.

21. The refrigeration device according to claim 11, characterized in that The sample carrier is a mesh covered with a membrane structure.

22. A refrigeration system comprising the refrigeration device according to any one of claims 7 to 21, characterized in that: include: The objective lens is used to observe the state of the sample at room temperature, the freezing process and the sample after freezing through the freezing substrate or the sample carrier.

23. The refrigeration system according to claim 22, wherein: The objective lens is an oil immersion objective lens.

24. The refrigeration system according to claim 23, wherein: The oil immersion objective lens is in contact with the frozen substrate; alternatively, the oil immersion objective lens is in direct contact with the sample carrier.

25. A method for manipulating a sample using the freezing system of claim 22, wherein: The steps include: Steps for freezing samples; The step of separating the frozen sample from the freezing device by using external force.

26. The method according to claim 25, characterized in that Before the step of freezing the sample, the method further comprises: Steps to control sample thickness by moving the sample carrier.

27. The method according to claim 25, characterized in that The step of freezing the sample comprises: adjusting electrical parameters of the heat source to maintain the average temperature of the sample at a first temperature; The electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the cold source can provide.

28. The method according to claim 27, characterized in that Before detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, the method further comprises: The sample is treated by increasing the temperature from the first temperature to the third temperature.

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

30. The method according to claim 27, wherein The first temperature is from 0° C. to 40° C., and the second temperature is lower than the critical temperature of the sample forming a glassy state.

31. The method according to claim 25, wherein Also includes: Observe the steps of freezing samples.

32. The method according to claim 25, wherein Also includes: The step of thawing the frozen sample by heating it with a heat source.

33. A method for manipulating a sample using the freezing system of claim 22, wherein: The steps include: placing the sample in the channel formed between the interface layer and the sample carrier; Steps for freezing samples; The step of separating the frozen sample from the freezing device by using external force.

34. The method according to claim 33, wherein The step of freezing the sample comprises: adjusting electrical parameters of the heat source to maintain the average temperature of the sample at a first temperature; The electrical parameter is detected and adjusted to a first predetermined range to adjust the average temperature of the sample to a second temperature, wherein the second temperature is lower than the first temperature, and the required temperature value is determined within the lowest temperature range that the cold source can provide.

35. The method according to claim 34, wherein Before detecting and adjusting the electrical parameter to a first predetermined range to adjust the average temperature of the sample to a second temperature, the method further comprises: The sample is treated by increasing the temperature from the first temperature to the third temperature.

36. The method according to claim 34, wherein The first temperature is the liquidus temperature of the sample, and the second temperature is the temperature at which the same sample is directly transformed from liquid to glassy state under the same environment and continuously maintains the glassy state.

37. The method according to claim 34, wherein The first temperature is from 0° C. to 40° C., and the second temperature is lower than the critical temperature of the sample forming a glassy state.

38. The method according to claim 33, wherein Also includes: Observe the steps of freezing samples.

39. The method according to claim 33, wherein Also includes: The step of thawing the frozen sample by heating it with a heat source.

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