Frozen sample deep etching and transferring device for low-temperature scanning probe microscope

Abstract

The invention discloses a frozen sample deep etching device for a low-temperature scanning probe microscope. The frozen sample deep etching device comprises vacuum equipment, a Dewar tank, a glove box, a heating pipe assembly, a sample conveying mechanism, a sample clamping and transferring mechanism and a sample conveyor, liquid nitrogen is contained in the Dewar tank, and the inner space of the glove box is filled with pure nitrogen volatilized from the liquid nitrogen; the vacuum equipment comprises a vacuum chamber body and a vacuum chamber cover, and the vacuum chamber body is located in the Dewar tank; the sample conveyor comprises a sample seat for accommodating a frozen sample to be deeply etched, the sample conveying mechanism is used for conveying the sample conveyor from the glove box to the inside of the Dewar tank, and the sample clamping and transferring mechanism is used for transferring the frozen sample in the sample conveyor from the Dewar tank to the inside of the vacuum chamber. The device can perform deep etching on a low-temperature scanning probe microscope sample and can perform low-temperature transfer on the etched sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sample preparation for scanning probe microscopes, and in particular to a deep etching device for cryogenic scanning probe microscopy. BACKGROUND

[0002] Scanning probe microscopes are microscopic imaging instruments that characterize the topography or other characteristic distribution of a sample surface using the interaction between a probe and the sample surface, including scanning tunneling microscopes, atomic force microscopes, magnetic force microscopes, etc. Cryogenic scanning probe microscopes are scanning probe microscopes that operate in a cryogenic environment and can characterize cryogenic biological samples or materials. For some special samples, such as biological tissues, cells, biological macromolecules, and electrophoresis gels, the microstructure strength is often lower than that of mineral crystals and other material samples, so when using mechanical scanning probe microscopy such as atomic force microscopy, these samples are often deformed or even damaged. However, if the temperature of these samples is lowered below their glass transition temperature, their microstructure strength will be greatly enhanced, so cryogenic scanning probe microscopes have unique advantages in characterizing the microstructure of such special samples.

[0003] In general, the surface of the sample provided to the cryogenic scanning probe microscope must be dry. If the sample surface contains water, the surface structure of the sample will be covered with ice after freezing and cannot be characterized. However, for biological samples or soft material samples and other special samples, their microstructure state is often most true in a solution environment. However, if we want to observe the structure of the sample in the solution under low temperature conditions, we need to perform deep etching on the sample under low temperature conditions, i.e. sublimate the ice on the surface of the sample to expose the sample to be characterized, so a device capable of deep etching of the sample for cryogenic scanning probe microscopes is needed.

[0004] The deep etching technology originated from the biological sample preparation of scanning electron microscope, and at present, the technology is still mainly applied to the scanning electron microscope imaging. The deep etching technology of the electron microscope sample includes deep etching and metal reformation film spraying, that is, a conductive film is deposited on the surface of the low-temperature sample by using the thin film deposition technology such as magnetron sputtering, so that the surface structure of the sample can be characterized by the scanning electron microscope. The technology needs to be completed by a special deep etching-reformation machine. However, although the deep etching-reformation machine contains etching function, the output sample is actually a replica after metal spraying reformation of the sample. The original low-temperature sample after etching cannot be directly obtained. On the other hand, the deep etching is similar to the freeze-drying technology, which is to leave the dried sample by sublimating the ice contained in the sample in a low-temperature vacuum state. However, the final output sample of the existing freeze-drying machine is also a dried normal-temperature sample. The dried sample at low temperature cannot be directly obtained, and the freeze-drying temperature of the freeze-drying machine is usually above-40 DEG C, which cannot maintain the low-temperature vitrification state of the biological sample. On the other hand, the cleanliness of the sample surface is crucial for scanning probe microscopy. The contaminants on the sample surface can seriously interfere with the imaging quality and mislead the analysis of the imaging results. Due to the cold trap effect, the low-temperature frozen sample is easy to adsorb contaminants in the environment higher than its own temperature. The existing freeze-drying equipment only considers the drying of the sample in the design, and its structure and use method cannot guarantee that the sample does not adsorb contaminants during the drying process.

[0005] In summary, there is currently a lack of a low-temperature deep etching device for scanning probe microscopy. Low-temperature quick freezing can preserve the original structure of biological samples such as proteins and some special material samples. If the low-temperature deep etching technology can preserve the biological samples or material samples in the low-temperature glass state and transfer the low-temperature samples to the low-temperature scanning probe microscope without warming and contamination, it will have great significance for the study of biological or material science. SUMMARY

[0006] The purpose of the present application is to provide a frozen sample deep etching device for low-temperature scanning probe microscopy, which can perform deep etching on the sample of the low-temperature scanning probe microscopy and can transfer the etched sample at low temperature.

[0007] The present application provides a frozen sample deep etching device for low-temperature scanning probe microscopy, comprising: a vacuum device, a dewar flask, a glove box, a heating pipe assembly, a sample transfer mechanism, a sample clamping and transferring mechanism, and a sample conveyor; wherein,

[0008] The dewar flask contains liquid nitrogen, the glove box is located at the mouth of the dewar flask, the glove box is in fluid communication with the dewar flask, and the internal space of the glove box is filled with pure nitrogen gas volatilized from the liquid nitrogen;

[0009] The vacuum device comprises a vacuum chamber body and a vacuum chamber cover, which jointly define a closed vacuum chamber, and the vacuum chamber body is located inside the dewar.

[0010] The sample conveyor comprises a sample seat for accommodating the frozen sample to be deeply etched, the sample conveying mechanism is used to convey the sample conveyor from the glove box to the inside of the dewar, and the sample gripping transfer mechanism is used to transfer the frozen sample in the sample conveyor from the dewar to the inside of the vacuum chamber.

[0011] The heating tube assembly is located inside the vacuum chamber and is used to heat the frozen sample, thereby completing the deep etching of the frozen sample.

[0012] In another preferred embodiment, the vacuum chamber body is located above the liquid nitrogen level in the dewar. In another preferred embodiment, the glove box is connected to the top of the dewar through a connecting flange.

[0013] In another preferred embodiment, the glove box is connected to the vacuum chamber body through a structural member, such as a connecting rod.

[0014] In another preferred embodiment, the bottom of the glove box is provided with a through hole.

[0015] In another preferred embodiment, the vacuum device further comprises a vacuum chamber cover and a first sealing member for sealingly connecting the vacuum chamber cover and the vacuum chamber body. Preferably, the first sealing member is a low-temperature generic seal ring, and more preferably, it comprises one low-temperature generic seal ring.

[0016] Preferably, the vacuum chamber cover can be opened and closed multiple times at a low temperature of -196°C while maintaining high sealing performance. Preferably, the vacuum chamber body comprises a protruding portion provided with the first sealing member, and the protruding portion is sealingly connected to the vacuum chamber cover through the first sealing member. Preferably, the vacuum chamber cover is of a cylindrical structure.

[0017] In another preferred embodiment, the top of the vacuum chamber cover is provided with a pressure lifter and a cantilever, the pressure lifter is a hollow structure, the pressure lifter comprises a hollow area, and the cantilever passes through the hollow area.

[0018] The frozen sample deep etching device further comprises a vacuum chamber cover operating rod connected to the cantilever, the vacuum chamber cover operating rod moves up and down along its axial direction, thereby driving the vacuum chamber cover to move up and down in the axial direction, and when the vacuum chamber cover operating rod rotates, it drives the vacuum chamber cover to make a translational motion in a plane perpendicular to the axial direction of the vacuum chamber cover operating rod.

[0019] In another preferred embodiment, the vacuum chamber lid lever is substantially perpendicular to the cantilever.

[0020] In another preferred embodiment, the vacuum chamber lid lever and the cantilever constitute an L-shaped component.

[0021] In another preferred embodiment, the vacuum chamber lid lever is fixedly connected to the cantilever.

[0022] In another preferred embodiment, the vacuum device further comprises a vacuum switch device, the vacuum switch device comprising a driving mechanism and a force transmission rod, the driving mechanism driving the force transmission rod to move up and down along its axial direction, the force transmission rod being configured to drive the vacuum chamber lid to move up and down along its axial direction when the force transmission rod moves up and down along its axial direction, thereby achieving the opening and closing of the vacuum chamber lid.

[0023] In another preferred embodiment, the upper surface of the pressure booster is provided with a positioning hole, and the lower end of the force transmission rod is provided with a latch, the latch cooperating with the positioning hole to form a locked state and an unlocked state of the force transmission rod and the pressure booster, when the force transmission rod and the pressure booster are in the locked state, the force transmission rod can drive the pressure booster to move upward along the axial direction of the force transmission rod, thereby providing a pulling force to open the vacuum chamber lid.

[0024] In another preferred embodiment, the positioning hole is composed of a waist-shaped hole and a circular hole.

[0025] In another preferred embodiment, the positioning hole is located above the hollow area of the pressure booster, and the force transmission rod can pass through the positioning hole into the hollow area. Preferably, the hollow area is located below the upper surface of the pressure booster.

[0026] In another preferred embodiment, the driving mechanism comprises a ball screw module, and the rotation of the ball screw of the ball screw module can drive the force transmission rod to move up and down along its axial direction.

[0027] In another preferred embodiment, the bottom of the force transmission rod is provided with a lens, and the vacuum chamber lid is provided with an observation window, and the lens can image the sample in the vacuum chamber through the observation window.

[0028] In another preferred embodiment, the sample delivery mechanism comprises a sample holder lever, the bottom of the sample holder lever being connected with a sample holder, the sample holder being located inside the dewar and outside the vacuum chamber, and the sample holder lever being configured to move up and down along its axial direction inside the dewar, thereby receiving the sample delivery device through the sample holder.

[0029] In another preferred embodiment, the sample carrier further comprises a carrier cover and a carrier base, and the sample holder is magnetically connected to the carrier cover and the carrier base.

[0030] In another preferred embodiment, an iron sheet is fixed on the upper surface of the sample holder, and a mica substrate is adhered to the iron sheet for carrying the biomacromolecule sample.

[0031] In another preferred embodiment, the cryogenic sample deep etching device further comprises a heat preservation sealer structure arranged outside the sample carrier, and the heat preservation sealer structure is used for heat preservation and sealing of the cryogenic sample during transfer of the cryogenic sample from the outside to the glove box.

[0032] In another preferred embodiment, the sample holder comprises a platform and a boss arranged in close proximity, the platform is provided with a platform circular groove, and the boss is provided with a boss circular groove for receiving a sample carrier, and the platform circular groove is used for receiving the carrier cover when the mechanical hand opens the sample carrier.

[0033] In another preferred embodiment, the heating tube assembly comprises a heat-resistant tube, a heating head, an electric heating rod, and a vacuum connector, one end of the heat-resistant tube is connected to the heating head, the other end is connected to the vacuum connector, the vacuum connector is used for fixedly and sealingly connecting the heating tube assembly and the vacuum chamber body, and the electric heating rod is arranged inside the heating head. Preferably, the cryogenic sample is placed above the heating head, and more preferably, a fixing member for fixing the cryogenic sample is arranged on the top of the heating head.

[0034] In another preferred embodiment, a rectangular groove is formed in the side edge of the heating head, and a thermocouple is fixed in the rectangular groove.

[0035] In another preferred embodiment, the electric heating rod is fixed at the center of the heating head.

[0036] In another preferred embodiment, the fixing member is a magnet.

[0037] In another preferred embodiment, the sample gripping and transferring mechanism comprises a mechanical hand and a mechanical hand operating lever, the mechanical hand operating lever comprises a torsion rod, the mechanical hand comprises a left jaw and a right jaw, and the left jaw and the right jaw are driven to open and close by the torsion rod to achieve gripping and releasing of the cryogenic sample.

[0038] In another preferred embodiment, one end of the torsion rod is provided with a push rod, and the push rod can drive the left jaw and the right jaw to open and close.

[0039] Preferably, the push rod is connected to the left jaw and the right jaw for transmitting the movement of the torsion rod to make the left jaw and the right jaw open.

[0040] In another preferred embodiment, a first elastic reset mechanism is arranged on the left clamping jaw, and a second elastic reset mechanism is arranged on the right clamping jaw, and the first elastic reset mechanism and the second elastic reset mechanism are configured to provide elastic energy storage when the left clamping jaw and the right clamping jaw are controlled by the mechanical arm control lever, and drive the left clamping jaw and the right clamping jaw to close through elastic rebound after the control is released.

[0041] In another preferred embodiment, the left clamping jaw is provided with a first tooth groove, the right clamping jaw is provided with a second tooth groove, and the shift rod is inserted into the first tooth groove and the second tooth groove respectively, and the shift rod drives the left clamping jaw and the right clamping jaw to open by rotating the torsion rod.

[0042] In another preferred embodiment, the left clamping jaw comprises a first clamping part and a first connecting part, and the right clamping jaw comprises a second clamping part and a second connecting part, and the first clamping part and the first connecting part form an L-shaped structure, and the second clamping part and the second connecting part form an L-shaped structure.

[0043] In another preferred embodiment, the first elastic reset mechanism comprises a first hollow groove arranged on the left clamping jaw and a first compression spring arranged in the first hollow groove, and / or the second elastic reset mechanism comprises a second hollow groove arranged on the right clamping jaw and a second compression spring arranged in the second hollow groove, and when the left clamping jaw and the right clamping jaw are opened, the first compression spring and the second compression spring are in a compressed state.

[0044] In another preferred embodiment, the first tooth groove is arranged on the first connecting part, and the second tooth groove is arranged on the second connecting part.

[0045] In another preferred embodiment, the torsion rod is substantially perpendicular to the first connecting part and the second connecting part.

[0046] In another preferred embodiment, the first clamping part comprises a first arc-shaped clamping part, and the second clamping part comprises a second arc-shaped clamping part, and when the left clamping jaw and the right clamping jaw are closed, the first arc-shaped clamping part and the second arc-shaped clamping part form a circle with a center A.

[0047] In another preferred embodiment, the center A of rotation of the circle coincides with the center B of the platform circular groove, the center C of the boss circular groove, and the center D of the heating tube.

[0048] In another preferred embodiment, the mechanical arm control lever, the vacuum chamber cover control lever, and the sample holder control lever are parallel.

[0049] It should be understood that, in the scope of the present application, the above technical features of the present application and the technical features specifically described in the following (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. It should be understood that the drawings described below are only some of the embodiments of the present application, and those skilled in the art can also obtain other embodiments from these drawings without creative labor.

[0051] Figure 1 is a schematic diagram of the whole structure of a cryogenic sample depth etching device for a low-temperature scanning probe microscope according to an embodiment of the present application;

[0052] Figure 2 is a sectional view of a vacuum chamber of a cryogenic sample depth etching device according to an embodiment of the present application;

[0053] Figure 3 is an assembly schematic diagram of a depth etching assembly of a cryogenic sample depth etching device according to an embodiment of the present application;

[0054] Figure 4 is a schematic diagram of a vacuum chamber and sample loading and unloading of a cryogenic sample depth etching device according to an embodiment of the present application;

[0055] Figure 5 is a schematic diagram of a heating tube structure of a cryogenic sample depth etching device according to an embodiment of the present application;

[0056] Figure 6 is a schematic diagram of the position relationship between a pressure booster and a force transmission rod according to an embodiment of the present application;

[0057] Figure 7 is a schematic diagram of a microscopic objective lens imaging light path according to an embodiment of the present application;

[0058] Figure 8 is a schematic diagram of a sample conveyor and its heat preservation sealer structure according to an embodiment of the present application;

[0059] Figure 9 is a schematic diagram of a sample holder structure according to an embodiment of the present application;

[0060] Figure 10 is a schematic diagram of the position relationship between a manipulator, a sample holder and a vacuum chamber according to an embodiment of the present application;

[0061] Figure 11is a schematic diagram of a booster structure according to an embodiment of the present application;

[0062] Figure 12 is a schematic diagram of a vacuum connector installation partial enlargement according to an embodiment of the present application;

[0063] Figure 13 is a schematic diagram of a sample seat structure according to an embodiment of the present application;

[0064] Figure 14 is a schematic diagram of a mechanical hand structure according to an embodiment of the present application;

[0065] Figure 15 is a schematic diagram of a vacuum chamber switch device structure according to an embodiment of the present application;

[0066] Figure 16 is a double-stranded DNA molecule atomic force microscopic image on a mica crystal surface according to an embodiment of the present application, wherein A is a result of scanning imaging by a low-temperature atomic force microscope after low-temperature etching of a sample frozen rapidly by a device of the present application, and B is a result of scanning imaging in a low-temperature atomic force microscope after blowing dry of a sample surface buffer by nitrogen;

[0067] Figure 17 is a cholera toxin B subunit protein molecule image on a mica surface according to an embodiment of the present application, wherein C is a sample subjected to low-temperature etching by a device of the present application, and D is a result of atomic force scanning microscopic imaging in a solution at normal temperature.

[0068] In each of the drawings, the following marks are used

[0069] 1-1 - vacuum chamber; 1-2 - dewar flask; 1-3 - glove box; 1-4 - evacuation tube; 1-5 - vacuum pump; 1-6 - touch screen; 1-7 - temperature controller; 1-8 - observation camera; 1-9 - glove box mounting hole; 1-10 - base plate; 1-13 - connecting rod; 2-1 - vent valve; 2-2 - pressure elevator; 2-3 - vacuum chamber evacuation tube; 2-4 - vacuum chamber cover; 2-5 - vacuum chamber vent tube; 2-6 - low temperature flooding seal ring; 2-7 - heating tube assembly; 2-8 - vacuum chamber body; 2-11 - coupling; 2-12 - vent valve switch rod; 2-13 - support; 2-14 - stainless steel bellows; 2-15 - stainless steel sleeve joint; 2-16 - reference surface; 2-17 - valve stem; 3-1 - manipulator control lever; 3-2 - sample holder control lever; 3-3 - pressure hand wheel; 3-4 - ball screw module; 3-5 - force transmission rod; 3-6 - glove box hatch; 3-8 - base plate of glove box; 3-9 - through hole; 3-10 - vacuum chamber cover control lever; 3-11 - tube seal; 3-12 - top plate of glove box; 4-1 - pressure head; 4-2 - lens; 4-3 - latch; 4-4 - positioning hole; 4-5 - cantilever; 4-6 - sample holder; 4-7 - manipulator; 4-8 - sample conveyor; 5-1 - heating head; 5-2 - thermocouple groove; 5-3 - thermocouple; 5-4 - magnet; 5-5 - heat resistant tube; 5-6 - vacuum connector; 5-7 - electric heating rod; 5-8 - low temperature epoxy resin; 5-9 - electrode; 5-10 - sealing groove; 5-11 - indium wire seal ring; 5-12 - bolt; 6-1 - observation hole; 6-2 - observation window; 6-3 - illumination optical fiber; 7-1 - infinite micro-lens group; 7-2 - tube lens; 8-1 - sample conveyor cover; 8-2 - sample seat; 8-3 - sample conveyor base; 8-4 - conical surface; 8-5 - upper magnet; 8-6 - lower magnet; 8-7 - bottom magnet; 8-8 - heat preservation sealing base; 8-9 - heat preservation sealing cover; 8-10 - screw thread; 8-11 - heat preservation sealer; 8-12 - fixing hole; 8-13 - conveyor cover V-shaped groove; 8-14 - sample seat V-shaped groove; 9-2 - platform; 9-3 - platform circular groove; 9-4 - boss; 9-5 - boss circular groove; 9-6 - boss magnet; 10-1 - manipulator track; 11-1 - waist-shaped hole; 11-2 - circular hole; 13-1 - iron sheet; 13-2 - mica substrate; 13-3 - biological macromolecule sample; 13-4 - sample seat upper magnet; 13-5 - sample seat lower magnet; 14-1 - left jaw; 14-2 - right jaw; 14-3 - upper cover plate; 14-4 - lower cover plate; 14-5 - fixing sleeve; 14-6 - thin-walled steel tube; 14-7 - sealer; 14-8 - torsion rod.14-9 – Torque handwheel; 14-10 – Hollow groove; 14-11 – Compression spring; 14-12 – Hollow groove; 14-13 – Compression spring; 14-14 – Gear groove; 14-15 – Gear groove; 14-16 – Lever; 14-17 – Washer; 14-18 – Screw; 15-1 – Lead screw; 15-2 – Slider; 15-3 – Force transmission rod caliper; 15-4 – Locking bolt; 15-5 – Locking handle. Detailed Implementation

[0070] Through extensive and in-depth research, the inventors have developed for the first time a cryogenic sample deep etching device for low-temperature scanning probe microscopy. The device includes a cryogenic Dewar flask, a glove box, a vacuum chamber, a vacuum chamber switching device, a robotic arm, a sample holder, a sample transfer device, and a thermal seal. The main advantage of this invention is that the vacuum chamber is located above the liquid nitrogen surface, and its inner wall temperature can be as low as liquid nitrogen temperature, thus creating a strong cold trap effect to adsorb contaminants during the etching process, ensuring the cleanliness of the sample surface. The use of a cryogenic sealing ring allows for a low-temperature, high-vacuum environment within the vacuum chamber, enabling etching of the sample at its glassy state temperature. Furthermore, the thermal seal and sample transfer device of this invention enable the transfer of biological samples at the glassy state temperature.

[0071] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0072] the term

[0073] As used herein, the terms “vent valve” and “sealing needle valve” are used interchangeably;

[0074] As used herein, the terms “vacuum chamber cover” and “vacuum chamber sealing cover” are used interchangeably; in this invention, all directional indicators (such as up, down, left, right, front, back, etc.) are used only to explain the relative positional relationship and movement of the components in a specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0075] A cryogenic sample depth etching device for low-temperature scanning probe microscopy

[0076] This invention discloses a cryogenic sample deep etching device for a low-temperature scanning probe microscope. The device enables low-temperature etching and transport of biological samples, comprising a vacuum unit, Dewar jars 1-2, a glove box 1-3, a heating tube assembly, a sample transfer mechanism, a sample gripping and transferring mechanism, and a sample conveyor 4-8.

[0077] The bottom of the cryogenic Dewar 1-2 is filled with liquid nitrogen. The glove box 1-3 is installed at the mouth of the cryogenic Dewar 1-2 and its bottom is connected to the inside of the Dewar 1-2. The interior space of the glove box 1-3 is filled with pure nitrogen gas evaporated from the liquid nitrogen. The vacuum chamber 1-1 is located above the liquid nitrogen level inside the Dewar 1-2 and is connected to the bottom plate of the glove box 1-3 through structural components.

[0078] The vacuum device includes a vacuum chamber body 2-8 and a vacuum chamber cover 2-4. The vacuum chamber body 2-8 and the vacuum chamber cover 2-4 together define a sealed vacuum chamber 1-1. The vacuum chamber body 2-8 is located inside the Dewar jar 1-2.

[0079] The sample conveyor 4-8 includes a sample holder 8-2 for accommodating a frozen sample to be deeply etched, a sample conveying mechanism for conveying the sample conveyor 4-8 from the glove box 1-3 to the inside of the Dewar jar 1-2, and a sample gripping and transferring mechanism for transferring the frozen sample in the sample conveyor 4-8 from the Dewar jar 1-2 to the inside of the vacuum chamber 1-1.

[0080] The heating tube assembly is located inside vacuum chamber 1-1. The heating tube assembly is used to heat the frozen sample, thereby completing the deep etching of the frozen sample.

[0081] Vacuum equipment

[0082] The vacuum equipment includes a vacuum chamber cover 2-4, a first sealing element for sealingly connecting the vacuum chamber cover 2-4 to the vacuum chamber body 2-8, a vacuum switch device, a ventilation assembly, and a pumping assembly;

[0083] The top of the vacuum chamber cover 2-4 is equipped with a pressure booster 2-2 and a cantilever 4-5. The pressure booster 2-2 has a hollow structure and includes a hollow area. The cantilever 4-5 passes through the hollow area.

[0084] The frozen sample deep etching device also includes a vacuum chamber cover control lever 3-10, which is connected to a cantilever 4-5. The vacuum chamber cover control lever 3-10 moves up and down along its axial direction, thereby driving the vacuum chamber cover 2-4 to move up and down in the axial direction. When the vacuum chamber cover control lever 3-10 rotates, it drives the vacuum chamber cover 2-4 to perform translational motion in a plane perpendicular to the axial direction of the vacuum chamber cover control lever 3-10.

[0085] The vacuum chamber 1-1 switching device includes a ball screw module and a force transmission rod 3-5. An infinity microscope objective lens is installed at the lower end of the force transmission rod 3-5, and a pin 4-3 is installed on the outside of the objective lens for opening the vacuum chamber sealing cover 2-4.

[0086] Preferably, the ventilation assembly includes a ventilation valve 2-1, and the extraction assembly includes an extraction pipe 1-4.

[0087] Preferably, the vacuum chamber cover 2-4 can be opened or closed by a control lever at a low temperature of around -196°C, and the sealing cover and vent valve have a sealing function at a low temperature of around -196°C.

[0088] Preferably, an observation window is installed in the middle of the vacuum chamber cover 2-4 to observe the etching progress of the sample.

[0089] Preferably, the device further includes a ventilation assembly for introducing air into the vacuum chamber 1-1.

[0090] Preferably, the device further includes a vacuum pumping assembly for evacuating the vacuum chamber 1-1.

[0091] Heating components

[0092] In one embodiment, the heating assembly includes a heating head 5-1, a magnet, a heating rod 5-7, a thermocouple 5-3, a heat-insulating tube 5-5, and a vacuum connector 5-6 (e.g., a mounting flange). One end of the heat-insulating tube 5-5 is connected to the mounting flange, and the other end is connected to the heating head 5-1 (e.g., a stainless steel column). The heating rod 5-7 is embedded in the center of the heating head 5-1, the magnet is embedded in the top of the stainless steel column, and the measuring head of the thermocouple 5-3 is also embedded in the top of the stainless steel column.

[0093] Sample transfer mechanism

[0094] In one embodiment, the sample transfer mechanism includes a sample holder lever 3-2, with a sample holder 4-6 connected to the bottom of the lever 3-2. The sample holder 4-6 is located inside the Dewar jar 1-2 and outside the vacuum chamber 1-1. The sample holder 4-6 is used to receive the sample transmitter 4-8. The lever 3-2 is configured to drive the sample holder 4-6 to move up and down inside the Dewar jar 1-2, thereby receiving the sample transmitter 4-8 from the glove box 1-3.

[0095] The sample transmitter 4-8 includes a transmitter cover 8-1, a sample holder 8-2, and a transmitter base 8-3. The sample holder 8-2 includes an upper sample holder magnet 13-4, a lower sample holder magnet 13-5, an iron sheet 13-1, and a substrate 13-2. The upper surface of the substrate 13-2 is adsorbed with a biomacromolecule sample 13-3.

[0096] Preferably, the upper end of the transmitter base 8-3 is a conical surface, and the inner side of the transmitter cover 8-1 (or sealing cover) is also a conical surface and forms a sealing fit with the conical surface of the transmitter base 8-3.

[0097] Preferably, the sample transmitter base 8-3 and the sample transmitter cover 8-1 are made of copper with high heat capacity. When the sample is encapsulated inside, the overall volume can greatly slow down the rate of sample temperature change.

[0098] Thermal insulation seal

[0099] The frozen sample deep etching device also includes a thermal insulation seal structure, which is disposed outside the sample conveyor 4-8. The thermal insulation seal structure is used to keep the frozen sample warm and sealed during the process of transferring the frozen sample from the outside to the glove box 1-3.

[0100] Optionally, the thermal seal 8-11 includes a thermal seal base 8-8 and a thermal seal cover 8-9. The thermal seal cover 8-9 and the thermal seal base 8-8 form a sealing fit through threads. The sample conveyor 4-8 can be fixed above the thermal seal base 8-8. The thermal seal base 8-8 has an annular groove on its upper part to reduce the contact area with the sample conveyor 4-8. The thermal seal cover 8-9 and the thermal seal base 8-8 are made of polytetrafluoroethylene with low thermal conductivity. During the sample transfer process, the thermal seal is exposed to ambient temperature. When the sample conveyor 4-8 containing the sample is encapsulated in the thermal seal 8-11, the rate of temperature change of the sample can be further slowed down.

[0101] Sample gripping and transfer mechanism

[0102] In one embodiment, the sample gripping and transfer mechanism includes a robotic arm 4-7 and a robotic arm control lever 3-1. The robotic arm control lever 3-1 includes a torsion bar. The robotic arm 4-7 includes a left jaw 14-1 and a right jaw 14-2. The jaws are located inside the cryogenic Dewar jar 1-2. The control lever is a hollow rod, with one end located at room temperature and the other end connected to the jaws. The torsion bar is located at the central axis of the control lever and is the same length as the control lever. One end of the torsion bar has a lever that can drive the left jaw 14-1 and the right jaw 14-2 of the robotic arm 4-7 to open and close, so as to realize the gripping and release of the frozen sample.

[0103] This application possesses at least one of the following advantages.

[0104] (a) The sample transfer device included in the present invention can be opened or closed by a robotic arm in the Dewar jar included in the present invention, and the sample transfer device is made of copper material with a large heat capacity, so the low temperature sample after etching can be transferred to cryogenic detection instruments such as cryo-atomic force microscope or cryo-electron microscope with basically no temperature rise and no pollution.

[0105] (b) The bottom of the Dewar jar described in this invention is filled with liquid nitrogen, and the area above the liquid surface is filled with clean nitrogen gas volatilized from the liquid nitrogen. The sample is exposed to the nitrogen environment before and after etching, thus completely isolating it from external pollution.

[0106] (c) The vacuum chamber included in this invention is located above the liquid nitrogen surface inside the Dewar jar, and its inner wall temperature is close to -196°C. Therefore, during the etching process, the inner wall becomes a cold trap to adsorb the vapor generated during the etching process, and the cold trap effect is better than that of existing deep etching machines or freeze dryers.

[0107] (d) The operation of the manipulator's gripper opening and closing, the sample conveyor opening and closing, the sample holder delivery, and the vacuum chamber cover opening and closing included in this invention can be completed by the operator by pushing, pulling or twisting the manipulator joystick, the vacuum chamber cover joystick and the sample joystick, which greatly reduces the complexity of operation.

[0108] (e) The cryogenic sample deep etching device for low-temperature scanning probe microscope of this application achieves the sealing of the vacuum chamber cover and the vacuum chamber body by setting up a vacuum operating rod, a force transmission rod, a vacuum chamber cover and a first sealing ring (preferably including two plug sealing rings), so that the vacuum chamber cover can maintain high sealing performance even after multiple opening and closing at a low temperature of -196°C.

[0109] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be understood that these are merely examples provided to the reader of possible implementations of the present invention and are not intended to limit the scope of the invention.

[0110] Example 1

[0111] like Figure 1 As shown, a low-temperature deep etching device for biological samples includes a vacuum chamber 1-1, a Dewar jar 1-2, a glove box 1-3, a vacuum tube 1-4, a molecular pump 1-5, a touch screen 1-6, a temperature controller 1-7, an observation camera 1-8, a base plate 1-10, and a connecting flange 1-11. The glove box 1-3 is made of transparent, colorless plexiglass, with glove mounting holes 1-9 on its left and front sides, a hatch 1-12 on its right side, and a connecting flange 1-11 at its bottom. The Dewar jar 1-2 is connected to the glove box 1-3 via the connecting flange 1-11. A connecting rod 1-13 is installed at the bottom of the glove box, and the vacuum chamber 1-1 is installed at the lower end of the connecting rod 1-13. When the glove box 1-3 is installed on the Dewar jar 1-2, the vacuum chamber 1-1 will be located inside the Dewar jar 1-2. Glove box 1-3 and Dewar jar 1-2 are mounted on base plate 1-10. Molecular pump 1-5 is also mounted on the left side of the Dewar jar, and is connected to vacuum chamber 1-1 via suction pipe 1-4. A monitoring camera 1-8 is mounted above glove box 1-3 to observe the depth etching progress, and a temperature controller 1-7 is also installed to control the sample temperature. A touch screen 1-4 is also mounted above base plate 1-10 to display the image captured by monitoring camera 1-8 and to set etching parameters.

[0112] like Figure 2 The diagram shows the structure of vacuum chamber 1-1. The right figure is an external view, and the left figure is a cross-sectional view along the reference plane 2-16, showing the vacuum chamber body 2-8, heating tube assembly 2-7, ultra-low temperature sealing ring 2-6, and vacuum chamber cover 2-4, with a pressure booster 2-2 installed at its top.

[0113] In this embodiment Figure 2 The image shows two low-temperature sealing rings 2-6. By setting two sealing rings, the sealing performance between the vacuum chamber cover 2-4 and the vacuum chamber body 2-8 can be enhanced.

[0114] A ventilation assembly and a vacuum pumping assembly are installed on the upper part of the vacuum chamber body 2-8. The ventilation assembly is used to introduce air into the vacuum body 2-8, and the vacuum pumping assembly is used to evacuate the vacuum body 2-8 into a vacuum. The vacuum pumping assembly includes a stainless steel vacuum chamber pumping pipe 2-3, a stainless steel bellows pipe 2-14, and a stainless steel compression fitting 2-15. The stainless steel vacuum chamber pumping pipe 2-3 and the stainless steel bellows pipe 2-14 are connected by the stainless steel compression fitting 2-15, which forms a seal with the stainless steel vacuum chamber pumping pipe 2-3 and the stainless steel bellows pipe 2-14. The other end of the bellows pipe 2-14 leads to the top of the glove box 1-3 and is connected to the pumping pipe 1-4 through a vacuum valve.

[0115] The ventilation assembly includes a stainless steel vacuum chamber ventilation pipe 2-5, a coupling 2-11, and a ventilation valve switch rod 2-12. The top of the vacuum chamber ventilation pipe 2-5 is equipped with a cryogenic sealing needle valve 2-1 (i.e., ventilation valve 2-1). The cryogenic sealing needle valve 2-1 includes a valve stem 2-17, which can control the opening and closing of the valve core. The upper end of the vent valve switch rod 2-12 leads to the top of the glove box 1-3, allowing the operator to easily switch the vent valve 2-1 outside the glove box 1-3. The coupling 2-11 is used to connect the vent valve switch rod 2-12 and the valve stem 2-17. By controlling the vent valve switch rod 2-12 to open the valve stem 2-17, the vent valve 2-1 is opened through the valve stem 2-17, which in turn opens the vacuum chamber vent pipe 2-5. The vent valve switch rod 2-12 is located above the valve stem 2-17, and the vacuum chamber vent pipe 2-5, the valve stem 2-17, and the vent valve switch rod 2-12 are on the same axis.

[0116] like Figure 5The diagram shows the specific structure of the heating tube assembly 2-7, which mainly includes a heating head 5-1, a heat-resistant tube 5-5, and a vacuum connector 5-6. A heating rod 5-7 is fixed to the center of the heating head 5-1. A rectangular groove 5-2 is formed on the side of the heating head 5-1, in which a thermocouple 5-3 is fixed. A magnet 5-4 is also fixed to the top of the heating head 5-1 for fixing the sample holder. The bottom end of the heat-resistant tube 5-5 is the vacuum connector 5-6, which serves two purposes: firstly, to seal and fix the heating tube assembly 2-7 to the vacuum chamber body 2-8; and secondly, to connect the internal and external wires of the vacuum chamber 1-1.

[0117] like Figure 12 The diagram shows the assembly structure of the heating tube assembly 2-7 and the vacuum chamber body 2-8. The bottom of the vacuum chamber body 2-8 has a sealing groove 5-10 containing an indium wire sealing ring 5-11. The vacuum connector 5-6 is fixed to the vacuum chamber body 2-8 by a set of bolts 5-12. The vacuum connector 5-6 forms a seal between itself and the vacuum chamber body 2-8 by pressing the indium wire. The center of the vacuum connector 5-6 is filled with low-temperature epoxy resin 5-8, into which four metal electrodes 5-9 are inserted. Both ends of the metal electrodes 5-9 are exposed outside the low-temperature epoxy resin 5-8, forming a seal between the electrodes 5-9 and the low-temperature epoxy resin 5-8. This also allows for the transmission of electrical signals inside and outside the vacuum chamber 1-1.

[0118] like Figure 3 The diagram shows the assembly of the main body of the deep etching device of the present invention. The bottom plate 3-8 of the glove box 1-3 serves as the support plate for the entire device, with a through hole 3-9 in its center. The bottom of the glove box is bolted to the connecting flange 1-11 of the Dewar canister, and a ball screw module 3-4 is mounted on it. A handwheel 3-3 is mounted on the top of the ball screw module 3-4, and a force transmission rod 3-5 is mounted on the slider of the ball screw module 3-4. A set of pipe sealing joints 3-11 is mounted on the top plate 3-12 of the glove box 1-3 to fix slender components such as the robot arm control lever 3-1, the sample holder control lever 3-2, and the vacuum chamber cover control lever 3-10. The robot arm control lever 3-1 is used to control the robot arm 4-7. The bottom end of the sample holder control lever 3-2 is connected to the sample holder 4-6. The movement of the sample holder control lever 3-2 drives the movement of the sample holder 4-6. For example, see [reference needed]. Figure 3 and Figure 4 The vacuum chamber cover control lever 3-10 is used to control the vacuum chamber cover 2-4 to move up and down along the axial direction of the vacuum chamber cover control lever 3-10, and to translate in a plane perpendicular to the axial direction of the vacuum chamber cover control lever 3-10.

[0119] like Figure 4The diagram shows the vacuum chamber and sample loading / unloading of the present invention. The vacuum chamber body 2-8 is connected to the stainless steel connecting rods 1-13 by screws. There are three stainless steel connecting rods 1-13 evenly distributed along the circumference, and they are fixed together by support members 2-13 to increase strength (see...). Figure 2 Stainless steel connecting rod 1-13 is used to fix the vacuum chamber body 2-8 to the glove box 1-3.

[0120] like Figure 4 and 6 The vacuum chamber cover 2-4 shown is topped with a pressure booster 4-4 and a cantilever 4-5 by screws. The pressure booster 4-4 is a hollow structure, and the cantilever 4-5 passes through the space in the middle of the pressure booster 2-2 (i.e., the hollow area in the middle), with its other end fixed to the vacuum chamber cover operating lever 3-10. Figure 6 The diagram shows the positional relationship between the pressure lifter and the force transmission rod 3-5. A lens 4-2 is installed at the bottom of the force transmission rod 3-5. When the vacuum chamber cover 2-4 is closed, the operator rotates the pressure handwheel 3-3 counterclockwise, causing the force transmission rod 3-5 to move downwards. The lens 4-2 is inserted into the positioning hole 4-4 of the pressure lifter 2-2 and enters the hollow area in the middle of the pressure lifter 2-2. The bottom platform of the force transmission rod 3-5 presses against the upper surface of the pressure lifter 2-2, causing the vacuum chamber cover 2-4 to press against the low-temperature sealing ring 2-6 and continue to slide downwards until the bottom of the vacuum chamber cover 2-4 contacts the vacuum chamber body 2-8, forming a seal between the vacuum chamber cover 2-4 and the vacuum chamber body 2-8. At this time, the lens 4-2 can image the sample surface located at the top of the heating head 5-1 through the observation window 6-2 on the vacuum chamber cover 2-4.

[0121] like Figure 7 The diagram shows the imaging optical path of the microscope objective of the present invention. The lens 4-2 includes an infinity microlens group 7-1. The light from the sample surface is collected by the lens group 7-1 and converted into parallel light. It is transmitted to the tube lens 7-2 through the internal space of the hollow pressure rod 4-1 and focused by the tube lens 7-2. The back focal plane of the tube lens 7-2 is located on the imaging chip surface of the imaging detector 1-8.

[0122] like Figure 8 The diagram shows the sample conveyor 4-8 and its heat-insulating seal structure of the present invention. The sample conveyor 4-8 includes a conveyor cover 8-1, a sample holder 8-2, and a conveyor base 8-3. The bottom and top of the conveyor base 8-3 respectively include an upper magnet 8-5 and a lower magnet 8-6. The contact surface 8-4 between the conveyor cover 8-1 and the conveyor base 8-3 is a conical surface. When the conveyor cover 8-1 and the conveyor base 8-3 are engaged, a self-sealing can be formed by the weight of the conveyor cover 8-1.

[0123] The thermal insulation seal 8-11 includes a transparent acrylic thermal insulation seal base 8-8 and a transparent acrylic thermal insulation seal cylinder 8-9. The thermal insulation seal base 8-8 includes a bottom magnet 8-7, which is used to fix the sample conveyor. The thermal insulation seal cylinder 8-9 and the thermal insulation seal base 8-8 are directly fixed and sealed by threads 8-10. Because the thermal insulation seal 8-11 is made of transparent acrylic glass with low thermal conductivity, the status of the sample conveyor 4-8 can be observed during sample transfer, and the rate of temperature rise can be slowed down.

[0124] like Figure 13 The diagram shows the specific structure of the sample holder 8-2. The upper and lower surfaces of the sample holder 8-2 are respectively inlaid with an upper magnet 13-4 and a lower magnet 13-5. The upper magnet attracts and fixes the iron sheet 13-1, to which a substrate 13-2 is adhered. A biomolecular sample 13-3 is adsorbed onto the upper surface of the substrate 13-2. The lower magnet 13-5 is used to fix the sample holder 8-2 to the sample transfer device base 8-3 or the heating head 5-1.

[0125] like Figure 9 As shown, the sample holder 4-6 includes a platform 9-2 and a boss 9-4. The platform 9-2 has a platform circular groove 9-3 in the middle. The boss 9-4 has a boss circular groove 9-5, and a boss magnet 9-6 is provided in the boss circular groove 9-5.

[0126] like Figure 14 The diagram shows the specific structure of the robotic arm 4-7 and the robotic arm control lever 3-1. The robotic arm 4-7 includes an upper cover plate 14-3, a left gripper 14-1, a right gripper 14-2, a lower cover plate 14-4, and a fixing sleeve 14-5. The upper cover plate 14-4, the grippers 14-1 and 14-2, and the lower cover plate 14-4 form a sandwich structure. The left gripper 14-1 and the right gripper 14-2 respectively contain hollow grooves 14-10 and 14-12, and the hollow grooves 14-10 and 14-12 contain pressure springs 14-11 and 14-13.

[0127] The tail ends of the left jaw 14-1 and the right jaw 14-2 are respectively provided with toothed grooves 14-14 and 14-15. The manipulator control lever 3-1 includes a thin-walled steel tube 14-6, a torsion bar 14-8, a sealer 14-7, and a torsion handwheel 14-9. The lower end of the torsion bar 14-8 is provided with a lever 14-16. The two ends of the lever 14-16 are inserted into the toothed grooves 14-14 and 14-15, respectively. By rotating the torsion handwheel 14-9 counterclockwise, the torsion bar 14-8 is rotated. The lever 14-16 at its bottom drives the left jaw 14-1 and the right jaw 14-2 to open to the left and right through the toothed grooves 14-14 and 14-15 to grasp the sample. At this time, the springs 14-11 and 14-13 are compressed. When the torque handwheel 14-9 is released, springs 14-11 and 14-13, through their rebound, drive the left jaw 14-1 and right jaw 14-2 to close, thus clamping the sample. When the torque lever is rotated, the lever 14-16 will cause the left and right jaws to move in opposite directions. At one end of each of the springs 14-11 and 14-13, there is a metal washer 14-17. This washer does not move with the jaws, thus compressing the springs.

[0128] like Figure 15 The diagram shows the specific structure of the vacuum chamber switching device of the present invention. The device includes a ball screw module 3-4, a pressure handwheel 3-3, a force transmission rod 3-5, a force transmission rod clamp 15-3, and a locking bolt 15-4. The ball screw module 3-4 includes a screw 15-1 and a slider 15-2. The force transmission rod clamp 15-3 is mounted on the slider 15-2. A C-shaped hole is machined on the force transmission rod clamp 15-3, through which the force transmission rod 3-5 passes. The rear end of the locking bolt 15-4 has a locking handle 15-5. When the locking bolt 15-4 is rotated clockwise, the C-shaped hole of the force transmission rod clamp 15-3 deforms, locking the force transmission rod 3-5. At this time, the force transmission rod 3-5 is fixed to the locking clamp 15-3.

[0129] When the locking bolt 15-4 is rotated counterclockwise, the C-shaped hole of the force transmission rod caliper 15-3 will release stress and eliminate deformation. At this time, the force transmission rod 3-5 can move axially within the C-shaped hole and can rotate freely.

[0130] The operational process of this application

[0131] When operating the system of this invention, the user must first add liquid nitrogen to the Dewar jar 1-2 and control the liquid nitrogen level to be below or slightly above the bottom of the vacuum chamber body 2-8. The operator must first encapsulate the sample using the sample transfer device 4-8 within the quick-freezing system and then seal the sample transfer device 4-8 using the thermal seal 8-11 to prevent frost formation on its surface and slow down the heating rate. Afterward, the sample transfer device 4-8 is transferred from the imaging system to the glove box 1-3. During the sample transfer, the thermal seal 8-11 can remain in a normal atmospheric environment. Because the liquid nitrogen in the Dewar jar 1-2 is constantly evaporating, the glove box 1-3 is filled with clean nitrogen. Therefore, after placing the entire thermal seal 8-11 into the glove box 1-3 and closing the glove box door 3-6, opening the thermal seal 8-11 inside the glove box 1-3 to expose the sample transfer device 4-8 to the nitrogen environment can prevent frost formation and contamination on its surface.

[0132] After the heat preservation seal 8-11 is opened, the operator pulls up the sample rack control lever 3-2 and uses tweezers to insert into the fixing hole 8-12 on the sample conveyor base 8-3 to grasp the entire sample conveyor 4-8. The boss 9-4 of the entire sample rack 4-6 is then inserted into the vent hole 3-9 on the glove box bottom plate 3-8, and the boss 9-4 of the sample rack 4-6 is exposed on the upper surface of the glove box bottom plate 3-8. Then, the entire sample conveyor 4-8 is placed on the boss 9-4 of the sample rack 4-6. The boss 9-4 has a boss circular groove 9-5, and a magnet 9-6 is located in the center of the groove. At this time, the magnet 9-6 on the boss will attract the magnet 8-6 on the sample conveyor base 8-3 and fix the entire sample conveyor 4-8. Then, the sample control lever 3-2 can be pushed down to convey the sample conveyor 4-8 into the Dewar jar 1-2.

[0133] Once the sample holder 4-6 and sample conveyor 4-8 have conveyed the sample into the Dewar jar 1-2, the robotic arm 4-7 can be operated to insert its jaws into the V-groove 8-13 of the sample conveyor cover 8-1 and pull upwards to open the sample conveyor cover 8-1. Figure 10The rotation trajectory 10-1 of the center A of the manipulator gripper shown can coincide with the center B of the circular groove 9-3 of the platform, the center C of the circular groove 9-5 of the boss, and the center D of the heating tube 2-7. Therefore, by rotating the manipulator lever 3-1, the sample conveyor cover 8-1 can be inserted into the circular groove 9-3 of the platform. Then, the operator rotates the manipulator lever 3-1 again to align the gripper with the sample holder 8-2. After that, the gripper is opened to engage with the V-shaped groove 8-14 of the sample holder. Then, it is pulled upward to separate the sample holder 8-2 from the conveyor base 8-3. Next, the manipulator is rotated to position the sample holder 8-2 above the heating tube assembly 2-7 (or the heating head 5-1). Then, the sample holder lever 3-1 is pushed downward to make the sample holder 8-2 contact the heating head 5-1. Finally, the manipulator gripper is released, and the sample holder 8-2 is fixed by the heating head 5-1.

[0134] After the sample holder 8-2 is placed on the heating tube assembly 2-7, the operator pulls the vacuum chamber cover operating lever 3-10 upwards so that the bottom surface of the vacuum chamber cover 2-4 is higher than the sample holder 8-2, and rotates the vacuum chamber cover 2-4 to the left so that it is directly above the vacuum chamber body 2-8. Then, the operator pushes the vacuum chamber cover operating lever 3-10 downwards so that the inner wall of the vacuum chamber cover 2-4 contacts the low-temperature plug seal ring 2-6. Afterwards, the operator pushes the force transmission lever 3-5 downwards so that the lens 4-2 at its bottom is positioned on the pressure lifter 2-2. Above hole 4-4, the positioning hole 4-4 is a combination of a circular hole 11-2 and an oblong hole 11-1. Observe the two pins on the outside of lens 4-2. Rotate the force transmission rod 3-5 so that pin 4-3 aligns with the oblong part of the positioning hole 4-4, and continue to push the force transmission rod 3-5 downwards so that pin 4-3 is fully inserted into the oblong hole, and the bottom stepped surface of the force transmission rod 3-5 contacts the upper surface of the pressure lifter 2-2. Then rotate the force transmission rod 3-5 about 90° to prepare for the next opening of the vacuum chamber cover 2-4. At this time, turn the handle on the slider of the ball screw module 3-4 to lock the force transmission rod 3-5, and turn the pressure handwheel 3-3 counterclockwise to push the vacuum chamber cover 2-4 downwards so that the low-temperature plug seal ring 2-6 enters the inner wall of the vacuum chamber cover 2-4. When you feel a significant increase in the rotational resistance of the pressure handwheel 3-3, it means that the vacuum chamber cover 2-4 has moved to the bottom, and the sealing between the vacuum chamber cover 2-4 and the vacuum chamber body 2-8 is completed.

[0135] At this point, the operator turns the vent valve handle clockwise to close the vent valve 2-1 and open the suction valve. Then, the vacuum pump can be started via the touch screen 1-6 to evacuate the vacuum chamber 1-1. When the vacuum level in the vacuum chamber 1-1 drops below 10 Pa, the deep etching program can be performed via the touch screen 1-6. At this time, the temperature controller 1-7 will power the heating rod 5-7 in the heating tube assembly 2-7. The heating rod 5-7 will then heat the heating head 5-1, which in turn will heat the sample holder 8-2 through heat transfer, ultimately achieving the purpose of heating the sample on the surface of the sample holder 8-2. The thermocouple 5-3 on the outside of the heating head 5-1 has its tip probe located near the junction of the sample holder 8-2 and the top of the heating head 5-1, close to the sample surface. The heating head 5-1 is in a vacuum environment. The heat-insulating tube 5-7 is made of thin-walled polyethylene. Since the heating head 5-1 is made of copper with a high heat capacity, its heat dissipation rate is very slow. Therefore, the sample surface temperature is basically the same as the temperature measured by the probe of the thermocouple 5-3, allowing the sample temperature to be well stabilized at the set temperature. The advantage of the vacuum chamber 1-1 design is that during the etching process, because the vacuum chamber 1-1 is in a high vacuum state, the saturated vapor pressure of water molecules is very low. The glassy water in the sample can sublimate at a temperature not exceeding the glass transition temperature of water, thus minimizing the damage of ice crystals to the sample. Another advantage of the vacuum chamber 1-1 of the present invention is that the entire vacuum chamber is located above the liquid nitrogen surface, and even the lower half of the vacuum chamber body 2-8 can be directly immersed in liquid nitrogen. Therefore, the inner wall temperature of the vacuum chamber 1-1 is extremely low, close to the temperature of liquid nitrogen, which can form a strong cold trap effect. If the gaseous substances generated during the etching process are not removed by the vacuum pump 1-5 in time, they can be quickly adsorbed by the inner wall of the vacuum chamber 1-1. Therefore, it can ensure that the sample is thoroughly etched and that the sample is clean.

[0136] Once the deep etching process begins, the surface condition of the sample is observed through the optical path formed by the microscope objective 7-1 and the tube lens 7-2, entering the detector 1-8 and being displayed on the touchscreen 1-6. After the operator observes the etching is complete, they can use the touchscreen to de-energize the heating rod 5-7 to stop etching. Then, they close the evacuation valve and shut off the vacuum pump to stop evacuation. Next, they open the vent valve 2-1, allowing the low-temperature clean nitrogen gas from the Dewar flask 1-2 to enter the vacuum chamber 1-1 through the vent valve 2-1 and vent pipe 2-3, balancing the internal and external pressures of the vacuum chamber and maintaining sample cleanliness. At this point, the operator can turn the pressure handwheel 3-3 clockwise, causing the force transmission rod 3-5 to move upwards. Since the force transmission rod 3-5 was rotated 90° before etching, the pin 4-3 is perpendicular to the oblong hole. Therefore, the pin 4-3 can move the pressure booster 2-2 upwards, opening the vacuum chamber cover 2-4. Next, pulling up the vacuum chamber cover lever and rotating it to the left will expose the etched sample back to the operator's view. The operator then uses the robotic arm 4-7 to place the sample holder 8-2 onto the sample transferor base 8-3 and seals the sample with the sample transferor cover 8-1. Then, pulling up the sample holder lever 3-2 will move the sample transferor 4-8 into the glove box 1-3. The sample transferor 4-8 is then sealed with the sample sealer 8-11, and finally, it can be transferred to the imaging equipment for imaging.

[0137] Example 2

[0138] This embodiment presents an example of experimental results obtained by scanning imaging using a low-temperature atomic force microscope after low-temperature etching using the cryogenic sample depth etching apparatus of this application.

[0139] Figure 16These are atomic force microscopy images of double-stranded DNA molecules on the surface of mica crystals. The actual diameter of the double-stranded DNA molecules is approximately 2 nm. The sample preparation method is as follows: a DNA solution is dropped onto the surface of a mica crystal and incubated for several minutes, allowing the DNA to deposit on the mica crystal surface. The DNA is fixed by the electrostatic interaction between its own surface and the mica. Then, the unfixed DNA is washed away with a buffer solution. Image A shows the result of scanning imaging using a low-temperature atomic force microscope after the sample has been flash-frozen and subjected to low-temperature etching according to this invention. Image B shows the result of scanning imaging using a low-temperature atomic force microscope after the buffer solution on the sample surface has been dried with nitrogen. In Figure A, the horizontal line A is perpendicular to a segment of DNA. The curve below shows the sample height distribution along the horizontal line A, with the highest point being the DNA apex, at a height of approximately 2 nm, almost equal to the actual diameter of the DNA molecule. Similarly, in Figure B, the curve below shows the sample height distribution along the horizontal line B, with the highest point at a height of approximately 1.3 nm, significantly smaller than the actual diameter of the DNA. This is because the sample in Figure B was dried by a nitrogen gas flow. Since nitrogen cannot completely remove all water molecules from the sample surface, the residual water molecule film creates surface tension that flattens the DNA molecule. Therefore, these experimental results demonstrate the advantage of the cryogenic sample depth etching device for low-temperature scanning probe microscopy proposed in this application, which helps low-temperature atomic force microscopy to restore the true structure of the sample.

[0140] Figure 17 The image shows a cholera toxin B subunit protein molecule on the surface of mica. The actual height of this molecule is approximately 2.5 nm. Image C shows the sample after low-temperature etching, prepared using the same method as the DNA sample. Image D shows the result of atomic force microscopy (AFM) imaging in solution at room temperature. The protein molecule crossed by line C has a height of approximately 2 nm, close to its actual height. The protein molecule crossed by line D has a height of approximately 1.5 nm, lower than the sample height reflected in the low-temperature AFM imaging results. Furthermore, the color intensity of the sample in the image also reflects its overall height; lighter colors indicate a higher height. It is evident that the overall height of the molecule in image C is significantly higher than the overall sample height in image D. Therefore, this experimental result demonstrates the advantage of the cryogenic sample depth etching device for low-temperature scanning probe microscopy combined with low-temperature AFM, compared to conventional room-temperature AFM, in reflecting the true structure of the sample more effectively.

[0141] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0142] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A device for deep etching of frozen samples in a low-temperature scanning probe microscope, characterized in that, include: Vacuum equipment, Dewar flasks (1-2), glove box (1-3), heating tube assembly, sample transfer mechanism, sample clamping and transfer mechanism, and sample conveyor (4-8); among which, The Dewar jar (1-2) is filled with liquid nitrogen. The glove box (1-3) is located at the mouth of the Dewar jar (1-2). The glove box (1-3) is in fluid communication with the Dewar jar (1-2). The interior space of the glove box (1-3) is filled with pure nitrogen gas evaporated from the liquid nitrogen. The vacuum device includes a vacuum chamber body (2-8) and a vacuum chamber cover (2-4), the vacuum chamber body (2-8) and the vacuum chamber cover (2-4) together define a sealed vacuum chamber (1-1), and the vacuum chamber body (2-8) is located inside the Dewar jar (1-2); The sample conveyor (4-8) includes a sample holder (8-2) for receiving the frozen sample to be deeply etched, the sample conveying mechanism is used to convey the sample conveyor (4-8) from the glove box (1-3) to the inside of the Dewar jar (1-2), and the sample gripping and transferring mechanism is used to transfer the frozen sample in the sample conveyor (4-8) from the Dewar jar (1-2) to the inside of the vacuum chamber (1-1); The heating tube assembly is located inside the vacuum chamber (1-1) and is used to heat the frozen sample to complete the deep etching of the frozen sample.

2. The frozen sample deep etching apparatus as described in claim 1, characterized in that, The vacuum device further includes a vacuum chamber cover (2-4) and a first sealing element, the first sealing element being used to seal the vacuum chamber cover (2-4) to the vacuum chamber body (2-8). Preferably, the first sealing element is a low-temperature plug seal ring (2-6), and more preferably, it includes two low-temperature plug seal rings (2-6).

3. The cryogenic sample deep etching apparatus as described in claim 2, characterized in that, The vacuum chamber cover (2-4) is provided with a pressure booster (2-2) and a cantilever (4-5) on the top. The pressure booster (2-2) is a hollow structure and includes a hollow area. The cantilever (4-5) passes through the hollow area. The frozen sample deep etching device also includes a vacuum chamber cover control lever (3-10), which is connected to the cantilever (4-5). The vacuum chamber cover control lever (3-10) moves up and down along its axial direction, thereby driving the vacuum chamber cover (2-4) to move up and down in the axial direction. When the vacuum chamber cover control lever (3-10) rotates, it drives the vacuum chamber cover (2-4) to perform translational motion in a plane perpendicular to the axial direction of the vacuum chamber cover control lever (3-10).

4. The cryogenic sample deep etching apparatus as described in claim 3, characterized in that, The vacuum equipment also includes a vacuum switch device, which includes a drive mechanism and a force transmission rod (3-5). The drive mechanism drives the force transmission rod (3-5) to move up and down along its axial direction. The force transmission rod (3-5) is configured to drive the vacuum chamber cover (2-4) to move up and down in its axial direction when the force transmission rod (3-5) moves up and down along its axial direction, thereby realizing the opening and closing of the vacuum chamber cover (2-4).

5. The cryogenic sample deep etching apparatus as described in claim 4, characterized in that, The upper surface of the pressure lifter (2-2) is provided with a positioning hole (4-4), and the lower end of the force transmission rod (3-5) is provided with a pin (4-3). The pin (4-3) cooperates with the positioning hole (4-4) to form a locked state and an unlocked state between the force transmission rod (3-5) and the pressure lifter (2-2). When the force transmission rod (3-5) and the pressure lifter (2-2) are in the locked state, the force transmission rod (3-5) can drive the pressure lifter (2-2) to move upward along the axial direction of the force transmission rod (3-5), thereby providing a pulling force to open the vacuum chamber cover (2-4).

6. The cryogenic sample deep etching apparatus as described in claim 4, characterized in that, A lens (4-2) is installed at the bottom of the force transmission rod (3-5), and an observation window (6-2) is provided on the vacuum chamber cover (2-4). The lens (4-2) can image the sample in the vacuum chamber (1-1) through the observation window (6-2).

7. The cryogenic sample deep etching apparatus as described in claim 1, characterized in that, The sample transfer mechanism includes a sample holder lever (3-2), the bottom of which is connected to a sample holder (4-6). The sample holder (4-6) is located inside the Dewar jar (1-2) and outside the vacuum chamber (1-1). The sample holder lever (3-2) is configured to move up and down along its axial direction inside the Dewar jar (1-2) to receive the sample transfer device (4-8) via the sample holder (4-6).

8. The frozen sample deep etching apparatus as described in claim 7, characterized in that, The sample conveyor (4-8) also includes a conveyor cover (8-1) and a conveyor base (8-3), and the sample holder (8-2) is magnetically connected to the conveyor cover (8-1) and the conveyor base (8-3).

9. The cryogenic sample deep etching apparatus as described in claim 1, characterized in that, The heating tube assembly (2-7) includes a heating head (5-1), a heat-insulating tube (5-5), a heating rod (5-7), and a vacuum connector (5-6). One end of the heat-insulating tube (5-5) is connected to the heating head (5-1), and the other end is connected to the vacuum connector (5-6). The vacuum connector (5-6) is used to fix and seal the heating tube assembly (2-7) to the vacuum chamber body (2-8). The heating rod (5-7) is disposed inside the heating head (5-1). Preferably, the frozen sample is placed above the heating head (5-1). More preferably, the top of the heating head (5-1) is provided with a fixing member for fixing the frozen sample.

10. The frozen sample deep etching apparatus as described in claim 1, characterized in that, The sample gripping and transfer mechanism includes a robotic arm (4-7) and a robotic arm control lever (3-1). The robotic arm control lever (3-1) includes a torsion bar (14-8), and the robotic arm (4-7) includes a left jaw (14-1) and a right jaw (14-2). The left jaw (14-1) and the right jaw (14-2) are driven to open and close by the torsion bar (14-8) to grasp and release the frozen sample.

Images