阅读说明：本技术 一种用于超声波植冰显微观察的装置及系统 (Device and system for ultrasonic ice-planting microscopic observation ) 是由 李维杰 彭正鑫 黄伟 漆琴 王义姚 刘宝林 于 2021-09-28 设计创作，主要内容包括：本发明公开了一种用于超声波植冰显微观察的装置及系统,属于细胞冻存领域,装置包括工作台以及置于工作台上的冷台,其中,冷台上设置有用于放置载玻片的载玻区,载玻区以及工作台上开设有轴向对齐的透光孔；工作台上还设置有用于为冷台提供冷量的冷源部件、用于为冷台提供热量的热源部件、用于检测冷台的温度的测温元件以及用于向冷台施加超声波的超声波振子。本发明结构简单且设计合理,能够方便地对生物组织冻存中的超声波植冰过程进行显微层面的观察。(The invention discloses a device and a system for ultrasonic ice implantation microscopic observation, belonging to the field of cell cryopreservation, wherein the device comprises a workbench and a cold platform arranged on the workbench, wherein a glass carrying area for placing a glass slide is arranged on the cold platform, and the glass carrying area and the workbench are provided with light holes aligned in the axial direction; the workbench is also provided with a cold source component for providing cold for the cold stage, a heat source component for providing heat for the cold stage, a temperature measuring element for detecting the temperature of the cold stage and an ultrasonic vibrator for applying ultrasonic waves to the cold stage. The ultrasonic ice-planting device is simple in structure and reasonable in design, and can conveniently observe the microscopic layer of the ultrasonic ice-planting process in the cryopreservation of biological tissues.)

1. A device for ultrasonic ice-planting microscopic observation is characterized in that: the glass slide cooling device comprises a workbench and a cooling table arranged on the workbench, wherein a glass carrying area for placing a glass slide is arranged on the cooling table, and light holes which are axially aligned are formed in the glass carrying area and the workbench; the workbench is also provided with a cold source component for providing cold for the cold stage, a heat source component for providing heat for the cold stage, a temperature measuring element for detecting the temperature of the cold stage and an ultrasonic vibrator for applying ultrasonic waves to the cold stage.

2. The apparatus for ultrasonic ice-planting microscopic observation according to claim 1, wherein: the cold source component is a cooling pipe used for conveying cooling liquid, and the heat source component is an electric heating wire.

3. The apparatus for ultrasonic ice-planting microscopic observation according to claim 2, wherein: the cooling pipe and the heating wire are arranged between the cold platform and the workbench or in the cold platform.

4. The apparatus for ultrasonic ice-planting microscopic observation according to claim 3, wherein: the surface of the working table is provided with a groove, and the cold table is arranged at the bottom of the groove.

5. The apparatus for ultrasonic ice-planting microscopic observation according to claim 4, wherein: the side of the workbench is provided with a liquid inlet joint and a liquid outlet joint which are communicated with the groove, and two ends of the cooling pipe are respectively connected to the liquid inlet joint and the liquid outlet joint.

6. The apparatus for ultrasonic ice-planting microscopic observation according to claim 4, wherein: when the cooling pipe is arranged between the cold platform and the workbench, the cooling pipe is half buried at the bottom of the groove.

7. The apparatus for ultrasonic ice-planting microscopic observation according to claim 4, wherein: the side surface of the workbench is provided with a connecting hole communicated with the groove, and the electric heating wire or a lead electrically connected with the electric heating wire penetrates through the connecting hole.

8. The apparatus for ultrasonic ice-planting microscopic observation according to claim 7, wherein: the temperature measuring element is a temperature sensor, and a probe of the temperature sensor extends into the groove from the outside of the workbench through the connecting hole.

9. The apparatus for ultrasonic ice-planting microscopic observation according to claim 1, wherein: the ultrasonic vibrator comprises a transducer and an amplitude transformer; the amplitude transformer is annular, the annular amplitude transformer is arranged outside the circumferential outer wall of the cold station, and the amplitude transformer is in contact with the circumferential outer wall of the cold station.

10. A system for ultrasonic ice-planting microscopic observation is characterized in that: comprising a microscope and a device according to any one of claims 1-9.

Technical Field

The invention relates to the technical field of cell cryopreservation, in particular to a device and a system for ultrasonic ice-planting microscopic observation.

Background

Cryopreservation of cells and tissues is a key technology for modern regenerative medicine, organ transplantation and assisted reproduction, and is widely concerned by all parties. The cryopreservation process of cells generally comprises the step of freezing the cells to a lower subzero temperature so as to realize long-term preservation, but the cells and tissues are damaged in the cryopreservation process: first, toxic damage from cryoprotectants; secondly, physical damage caused by ice crystal formation in cells by programmed cooling.

At present, cell freezing of a biological sample library is carried out by adopting a program cooling method, but in the traditional program cooling process, the solution is still not frozen when the temperature of the solution is lower than the eutectic temperature, so that the cell solution is supercooled, and sudden nucleation can be carried out when the supercooling degree is larger, so that the ice crystals are larger and physical damage is caused. The ice planting is that the temperature is kept constant near the eutectic point of the cell solution for a certain time, then the extracellular solution is firstly made into eutectic by using the methods of adding ice nucleus, touching the outer wall of the freezing storage tube by frozen metal, applying ultrasonic waves and the like, the concentration of the uncrystallized protective solution is increased because the dilute solution is preferentially crystallized in the growth process of the eutectic at the temperature, and the cells are gradually dehydrated under the action of osmotic pressure, so that the formation of the intracellular ice can be prevented or reduced, and the physical damage is reduced. Researchers find that the survival rate of cells can be effectively improved by adopting a certain ice planting operation in the process of programmed cooling, and the ultrasonic ice planting becomes the main research direction of the ice planting method and has a positive effect on the preservation of biological samples because the ultrasonic waves can reduce the supercooling degree and improve the nucleation temperature by utilizing the cavitation effect, the ice planting effect is obvious and the operation is simple and convenient.

In order to deeply research the ultrasonic ice-planting, researchers begin to pay more attention to the ultrasonic nucleation mechanism and the influence of the ultrasonic nucleation mechanism on cell tissues in the cryopreservation process, but the pressure of cavitation bubbles, bubbles and the like are under the microscopic condition, and the change process of the cavitation bubbles is very short, so that the observation needs to be carried out by matching with a microscope. Therefore, it is necessary to observe the ultrasonic ice-planting in real time at a microscopic scale, and an observation instrument capable of continuously observing the microscopic change of the histiocyte in the ultrasonic ice-planting process in real time at a low temperature does not exist at present.

Disclosure of Invention

Aiming at the problem that a microscope in the prior art cannot observe a micro tube in the ultrasonic ice-planting process, the invention aims to provide a device and a system for ultrasonic ice-planting microscopic observation.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, the invention provides a device for ultrasonic ice implantation microscopic observation, which comprises a workbench and a cold table arranged on the workbench, wherein a glass carrying area for placing a glass slide is arranged on the cold table, and the glass carrying area and the workbench are provided with light holes aligned in the axial direction; the workbench is also provided with a cold source component for providing cold for the cold stage, a heat source component for providing heat for the cold stage, a temperature measuring element for detecting the temperature of the cold stage and an ultrasonic vibrator for applying ultrasonic waves to the cold stage.

Preferably, the cold source component is a cooling pipe for conveying cooling liquid, and the heat source component is an electric heating wire.

Preferably, the cooling pipe and the heating wire are both arranged between the cooling table and the workbench or inside the cooling table.

Preferably, the surface of the working table is provided with a groove, and the cold table is arranged at the bottom of the groove.

Preferably, the side of the workbench is provided with a liquid inlet joint and a liquid outlet joint which are communicated with the groove, and two ends of the cooling pipe are respectively connected to the liquid inlet joint and the liquid outlet joint.

Preferably, when the cooling pipe is arranged between the cooling table and the working table, the cooling pipe is half buried in the groove bottom of the groove.

Preferably, a connection hole communicated with the groove is formed in the side surface of the workbench, and the electric heating wire or a lead electrically connected with the electric heating wire is arranged in the connection hole in a penetrating manner.

Preferably, the temperature measuring element is a temperature sensor, and a probe of the temperature sensor extends into the groove from the outside of the workbench through the connecting hole.

Preferably, the ultrasonic vibrator comprises a transducer and an amplitude transformer; the amplitude transformer is annular, the annular amplitude transformer is arranged outside the circumferential outer wall of the cold station, and the amplitude transformer is in contact with the circumferential outer wall of the cold station.

In another aspect, the present invention also provides a system for ultrasonic ice-implantation microscopy, comprising a microscope and a device as described above.

Adopt above-mentioned technical scheme, owing to be used for placing the glass section of slide glass on the cold stage, be used for providing cold volume cold source part for the cold stage on the slide glass same as the light trap of logical light and the workstation, be used for providing the heat source part of heat for the cold stage, a temperature element for detecting the temperature of cold stage, a setting for applying ultrasonic wave oscillator of ultrasonic wave to the cold stage, make to install the back on the objective table of microscope, can maintain the temperature that slide glass was carried on the cold stage at the temperature that is suitable for carrying out the ultrasonic wave ice planting through mutually supporting of cold source part, heat source part, temperature element, rethread ultrasonic wave oscillator is applied the ultrasonic wave and can be implemented the ice planting process, and then make ice planting process accessible microscope implement the observation.

Drawings

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a top view of the present invention;

FIG. 3 is a top view of the present invention with the cold plate removed.

In the figure, 1-workbench, 11-groove, 12-liquid inlet joint, 13-liquid outlet joint, 2-cold stage, 3-cold source component, 4-heat source component, 5-temperature measuring element, 6-ultrasonic vibrator, 61-transducer, 62-amplitude transformer, 7-light hole and 8-hole connecting pipe.

Detailed Description

The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

It should be noted that in the description of the present invention, the terms "upper", "lower", "left", "right", "front", "rear", and the like indicate orientations or positional relationships based on structures shown in the drawings, and are only used for convenience in describing the present invention, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

In the technical scheme, the terms "first" and "second" are only used for referring to the same or similar structures or corresponding structures with similar functions, and are not used for ranking the importance of the structures, or comparing the sizes or other meanings.

In addition, unless expressly stated or limited otherwise, the terms "mounted" and "connected" are to be construed broadly, e.g., the connection may be a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; the two structures can be directly connected or indirectly connected through an intermediate medium, and the two structures can be communicated with each other. To those skilled in the art, the specific meanings of the above terms in the present invention can be understood in light of the present general concepts, in connection with the specific context of the scheme.

Example one

The utility model provides a device for ultrasonic wave ice implantation microscopic observation, as shown in fig. 1-3, includes workstation 1 and arranges cold platform 2 on workstation 1 in, wherein, is provided with the glass slide district that is used for placing the slide glass on cold platform 2, sets up axially aligned light trap 7 on glass slide district and the workstation 1. The table 1 is further provided with a cold source member 3 for supplying cold to the cold stage 2, a heat source member 4 for supplying heat to the cold stage 2, a temperature measuring element 5 for detecting the temperature of the cold stage 2, and an ultrasonic transducer 6 for applying ultrasonic waves to the cold stage 2.

In the present embodiment, the table 1 is preferably made of a heat insulating material, the table 1 is configured in a rectangular flat plate structure so as to be placed on the stage of the microscope in use, the upper surface of the table 1 is provided with a sunken groove 11, the above-mentioned cold stage 2 is placed on the bottom of the groove 11, and the thickness of the cold stage 2 is configured so that the top surface of the cold stage 2 is flush with the upper surface of the table 1 when the cold stage 2 is placed in the groove 11, and further, it is preferable that the cold stage 2 is made of a good thermal conductor, such as a silver cold stage 2. In this embodiment, configuration cold platform 2 is discoid and its top surface is for carrying the glass district, and the cross section of corresponding recess 11 also configures into circularly to set up foretell light trap 7 on the axis of cold platform 2 and the axis of recess 11, place cold platform 2 during the use and locate the center department of recess 11 and can make light trap 7 on cold platform 2 and the light trap 7 axial alignment on the workstation 1, thereby make light can follow workstation 1 lower surface one side shine on the slide glass that is located cold platform 2 top surface one side.

In this embodiment, the cold source component 3 is configured as a cooling pipe for conveying a cooling liquid, preferably liquid nitrogen, and the cooling pipe is installed in the groove 11, and the cooling pipe is disposed between the cold stage 2 and the working table 1, that is, the cooling pipe is disposed at the bottom of the groove 11, and the cold stage 2 covers the cooling pipe when in use. In the embodiment, a clamping groove for clamping and embedding the cooling pipe is further formed in the bottom of the groove 11, so that the cooling pipe 3 is half buried in the bottom of the groove 11. Furthermore, the side face of the workbench 1 is provided with a liquid inlet joint 12 and a liquid outlet joint 13 which are communicated with the groove 11, and the two ends of the cooling pipe are respectively connected to the liquid inlet joint 12 and the liquid outlet joint 13, so that the flatness and tidiness of the top surface of the device are guaranteed, the convenience of the device is improved, the device can move along with the microscope objective table, and only joints on pipelines for conveying liquid nitrogen are connected to the liquid inlet joint 12 and the liquid outlet joint 13 during use.

It is understood that, in order to increase the contact area between the cold source part 3 and the cold stage 2, in one embodiment, the cooling pipe may be configured to be flat and/or wound in a disk shape.

In this embodiment, the heat source component 4 is configured as a heating wire, the heating wire is also disposed between the cooling platform 2 and the working platform 1, that is, the heating wire is also laid on the bottom of the groove 11, and usually, the heating wire is wrapped with a heat conducting and electric insulating material. In this embodiment, a hole communicating with the groove 11 is further formed in the side surface of the table 1, so that the heating wire or a wire electrically connected to the heating wire is inserted into the hole. And further, a hole connecting pipe 8 is sleeved in the hole, and two ends of the hole connecting pipe 8 respectively extend towards the inside of the groove 11 and the outside of the workbench 1, so that protection is provided for the heating wire or the conducting wire.

In this embodiment, the temperature measuring element 5 is disposed as a temperature sensor, and a probe of the temperature sensor is inserted into the recess 11 from the outside of the table 1 through the above-mentioned connection hole (via pipe 8). For example, a platinum resistance temperature sensor is arranged, a probe of the platinum resistance temperature sensor is in good close contact with the cooling stage 2, for example, a temperature measuring hole is formed in the side wall of the cooling stage 2, and the probe of the temperature sensor extends into the temperature measuring hole when in use, so that more accurate temperature information inside the cooling stage 2 is acquired.

In the present embodiment, the ultrasonic vibrator 6 includes a transducer 61 and a horn 62, and the horn 62 is preferably annular, and the annular horn 62 is disposed outside the circumferential outer wall of the cold stage 2. Wherein, the transducer 61 is fixedly arranged at the bottom of the groove 11, the amplitude transformer 62 is connected with the transducer 61, the amplitude transformer 62 surrounds the circumferential side wall of the cold stage 2, and is contacted with the circumferential outer part of the cold stage 2 through a conduction piece made of rigid material, the conduction piece is preferably a plurality of and is circumferentially and uniformly arranged, so that the ultrasonic wave can be conducted to the cold stage 2 and further transmitted to the glass slide, and the biological sample in the glass slide is subjected to ice planting. In another embodiment, the horn 62 is not in direct contact with the cold stage 2, and the horn 62 is configured to be annular and have a focal point, such as an annular configuration of two parallel cuts from a spherical surface, where the center of the sphere is the focal point. During the use, only need make the focus of amplitude transformer 62 aim at the slide glass can, the ultrasonic wave can pass through the air and transmit the slide glass, and then plant ice to the biological sample in the slide glass.

It can be understood that, in another embodiment, in order to further provide the contact area between the cold source component 3 and the cold stage 2, a mounting hole is provided inside the cold stage 2, and the cooling pipe constituting the cold source component 3 is inserted into the mounting hole, so that the whole area of the circumferential side wall of the cooling pipe can release cold to the cold stage 2, thereby improving the refrigeration effect. Accordingly, the heating wire constituting the heat source member 4 is also embedded in the cooling stage 2, thereby improving the heating effect.

When in use, the workbench 1 is placed on the objective table of a microscope, a glass slide containing a biological sample is placed on the cold stage 2, a delivery pipe of liquid nitrogen is connected to the liquid inlet joint 12, a return pipe of the liquid nitrogen is connected to the liquid outlet joint 13, and the electric heating wire is connected to a power supply and the temperature sensor is connected to a display or control device; the cooling rate of cold platform 2 can be controlled through the velocity of flow of control liquid nitrogen and the power of heating wire, when reaching the plant ice temperature, has the power of own heating wire to maintain the temperature of planting ice through the velocity of flow that changes the liquid nitrogen, starts ultrasonic vibrator 6 and plants the ice operation to the biological sample, can observe the phenomenon of planting the ice in-process this moment through the microscope.

In another embodiment, the cold source component 3 is configured as a semiconductor refrigeration sheet, one side of the cold end of the semiconductor refrigeration sheet is in contact with the circumferential side wall of the cold stage 2, one side of the hot end of the semiconductor refrigeration sheet faces the side wall of the groove 11, and a notch opposite to the hot end of the semiconductor refrigeration sheet is formed in the side surface of the workbench 1 and used for installing a radiator. Or in another embodiment, the cold source component 3 is configured as a stirling cooler, and the cold head of the stirling cooler is connected to the cold plate 2.

Example two

A system for ultrasonic ice-planting microscopic observation comprises a microscope and the device disclosed in the first embodiment, wherein the workbench 1 is placed on the object stage of the microscope. The system usually further comprises a display device, such as a display screen, electrically connected to the temperature measuring element 5 for displaying the temperature value. The system also comprises an ultrasonic generator which is matched with the ultrasonic vibrator 6 for use, wherein the ultrasonic generator is connected with the mains supply through a lead and is used for converting the mains supply into a high-frequency alternating current signal matched with the energy converter 61 so as to drive the energy converter 61 to work. The system also comprises a liquid nitrogen storage tank and a liquid nitrogen pump which are used for providing liquid nitrogen for the cooling pipe, and a control valve is arranged on a liquid conveying pipe connected between the outlet of the liquid nitrogen pump and the liquid inlet joint 12 so as to conveniently regulate and control the flow of the liquid nitrogen. And the heating wire is provided with a current regulator for regulating the current of the heating wire.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.