WO2020223860A1

WO2020223860A1 - Laser pulse thawing system for automated non-contact biological samples

Info

Publication number: WO2020223860A1
Application number: PCT/CN2019/085565
Authority: WO
Inventors: 金波; 刘湘娟; 王伟
Original assignee: 力盟生命科技(深圳)有限公司; 力盟低温医学(深圳)有限公司
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2020-11-12

Abstract

Disclosed is a laser pulse thawing system for automated non-contact biological samples, comprising: a main control module (1), a laser pulse generation subsystem (2), a sample identification subsystem (3) and a sample transmission subsystem (4). The main control module (1) is used for controlling the laser pulse generation subsystem (2), the sample identification subsystem (3) and the sample transmission subsystem (4) respectively and enabling a cooperative work among the laser pulse generation subsystem (2), the sample identification subsystem (3) and the sample transmission subsystem (4). The sample identification subsystem (3), the laser pulse generation subsystem (2) and the sample transmission subsystem (4) work cooperatively to complete rapid automated non-contact thawing and recording of the samples; and the new technology of laser thawing is applied to actual production, the whole process from sample removal to thawing recovery can be automatically completed to make the process control precise and improve the working efficiency.

Description

Laser pulse thawing system for automatic non-contact biological samples

Technical field

The invention relates to the technical field of biological engineering, in particular to a laser pulse thawing system for automatic non-contact biological samples.

Background technique

Assisted reproductive technology is a comprehensive technology integrating embryo division, mosaicism, transplantation, in vitro fertilization, genetic modification and other technologies. It is not only an important technological revolution in the field of animal reproduction, but also an important technical means to treat human infertility. Using this technology can fully excavate the genetic and reproductive capacity of animals, increase the number of offspring of purebred livestock and outstanding individuals, shorten the improvement cycle of livestock, and preserve embryos and genes with excellent genetic characteristics. It is widely used in the fields of biology and medicine. Research and application.

Among them, there are two main ways to preserve eggs and embryos: one is programmed freezing technology, and the other is vitrification preservation technology. The programmed preservation technology is the process of slow cooling through the program cooling instrument under the control of the computer program. The general cooling speed is 0.5-1 degrees/minute. The disadvantage of this method is that it requires a programmed cooling instrument and takes 2-3 hours. The advantage is The cryoprotectant has a relatively low concentration and is less toxic. The disadvantage of vitrification preservation technology is that high-concentration cryoprotectants may be toxic. The advantage is that the entire process is very short, sample handling is simple, and the survival rate is relatively high.

At present, vitrification preservation technology has become the mainstream choice for frozen samples, but this method has very strict requirements on the temperature, time and process of processing samples. The survival rate obtained by this method is very sensitive to the thawing rate of frozen samples. The thawing rate is the most important factor affecting the high survival rate of cells. Jin Bo et al.'s method of thawing eggs and embryos using laser pulses is a new fast thawing technology. This new technology greatly increases the rate of defrosting and rewarming through the energy of laser pulses, thereby reducing the concentration of cryoprotectants and reducing processing Sample time, this new technology has the advantages of both programmed and vitrified biological sample preservation methods, while overcoming their respective shortcomings.

technical problem

At present, the laser pulse thawing method usually uses manual control of the sample and laser pulse device to perform the thawing operation. It cannot be applied to actual production. There is no device that can automatically and accurately control the sample and laser pulse excitation. Therefore, it is necessary to study an automation The laser pulse thawing equipment can accurately locate the position of the cells and control the laser pulse not to contact the eggs and embryos, eliminating the possible impact on the development of the eggs and embryos because the laser directly contacts the biological samples.

Technical solutions

In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an automated non-contact laser pulse thawing system for biological samples.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

An automatic laser pulse thawing system for non-contact biological samples, which includes a main control module, a laser pulse generation subsystem, a sample recognition subsystem, and a sample transmission subsystem;

The main control module is used to control the laser pulse generation subsystem, the sample recognition subsystem, and the sample transmission subsystem respectively, and make the laser pulse generation subsystem, the sample recognition subsystem, and the sample transmission subsystem work together;

The laser pulse generation subsystem is mainly used to generate laser pulses under the control of the main control module. The laser pulse generation subsystem includes a laser trajectory control module for controlling the trajectory of the laser pulse and a laser for controlling the emission of the laser pulse. A transmitting module, the laser trajectory control module and the laser transmitting module are respectively controlled by the main control module;

The sample recognition subsystem is mainly used to quickly identify, locate and record the laser thawing process of samples;

The sample transmission subsystem is mainly used to transport the sample to the thawing position, to control the sample to leave the liquid nitrogen quickly, to control the sample to be immersed in the thawing liquid, and to transport the sample back to the initial position after thawing.

Preferably, the sample transmission subsystem includes a sample fixing device and a sample transmission device, a liquid nitrogen transmission device, and a thawing solution transmission device controlled by the main control module; the sample fixing device is used to fix the sample, and the sample transmission The device is used to transport the sample from the initial position to the thawing position and return the sample to the initial position after thawing. The liquid nitrogen transmission device is used to control the rapid separation of the sample and the liquid nitrogen to facilitate the laser pulse generation subsystem to perform laser thawing. The thawing liquid transmission device is used to move the thawing liquid to the vicinity of the sample after laser thawing, so that the sample is quickly immersed in the thawing liquid.

Preferably, a liquid nitrogen box for containing liquid nitrogen is provided in the liquid nitrogen transmission device, and a clamping device for clamping and fixing the rod cap is provided in the liquid nitrogen box.

Preferably, a laser energy control module and a laser pulse width control module are integrated in the main control module.

Preferably, it also includes a sample optical observation module, the sample optical observation module is set in the initial position for optical magnification observation of the thawed sample.

Preferably, it also includes a user interaction module, which is electrically connected to the main control module for realizing control of the entire system through the main control module.

Preferably, the user interaction module includes a display, a touch screen, a mouse, and a keyboard that are electrically connected to the main control module, respectively.

Preferably, it further includes a shared optical path system, the laser pulse generation subsystem acts on the sample through the shared optical path system, and the sample recognition subsystem performs sample recognition through the shared optical path system and records the laser thawing process.

Preferably, it also includes a power supply module, which is used to supply power to the main control module, the laser pulse generation subsystem, the sample identification subsystem, and the sample transmission subsystem respectively.

Beneficial effect

The present invention presets the empirical parameters of laser energy, pulse width and trajectory through the main control module and the laser pulse generation subsystem, and uses the collaborative work of the sample recognition subsystem, the laser pulse generation subsystem and the sample transmission subsystem to complete the automation of the sample Non-contact rapid thawing and recording, using the characteristics of high energy density of lasers to increase the thawing rate of frozen biological samples, and realize ultra-fast thawing of biological samples, thereby greatly improving the survival rate and development potential of biological samples; This device can apply the new technology of laser thawing in actual production, and can automatically complete the entire process from sample removal to thawing and recovery, which not only makes the process control accurate, but also improves work efficiency; at the same time, the system is more difficult to preserve The freezing and thawing of embryos and eggs has very good effects, which is of great significance for the research and popularization of embryo and egg freezing methods.

Description of the drawings

Figure 1 is a block diagram of the system structure of an embodiment of the present invention;

Fig. 2 is a schematic diagram of the structure of the carrier rod in the embodiment of the present invention.

Embodiments of the invention

Detailed ways

The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

As shown in Figure 1, the embodiment of the present invention provides an automated non-contact laser pulse thawing system for biological samples, which includes a main control module 1, a laser pulse generation subsystem 2, a sample recognition subsystem 3, and a sample transmission subsystem 4 ；

The main control module 1 is mainly used to control the laser pulse generation subsystem 2, the sample recognition subsystem 3 and the sample transmission subsystem 4 respectively, and make the laser pulse generation subsystem 2, the sample recognition subsystem 3 and the sample transmission subsystem 4 work together. ；

The laser pulse generation subsystem 2 is mainly used to generate laser pulses under the control of the main control module 1. The laser pulse generation subsystem 2 includes a laser trajectory control module 21 for controlling the trajectory of the laser pulse and a laser for controlling the emission of the laser pulse. The emission module 22, the laser track control module 21 and the laser emission module 22 are respectively controlled by the main control module 1;

The sample recognition subsystem 3 is mainly used to quickly identify, locate and record the laser thawing process of samples;

The sample transmission subsystem 4 is mainly used to transport the sample to the thawing position, control the sample to leave the liquid nitrogen quickly, control the sample to be immersed in the thawing liquid, and transport the sample back to the initial position after thawing.

Based on the above structural settings, before thawing the sample, it is necessary to preset the empirical parameters of the laser trajectory through the main control module 1 and the laser trajectory control module 21 according to the type and size of the sample; the sample is usually placed at the end of the carrier, and each sample The initial position is basically the same, and further calibration is required through the sample recognition subsystem 3 to complete accurate thawing. At the same time, the sample recognition subsystem 3 completes the recording of the thawing process, so that the operator can confirm and archive the thawing process and effects; The transmission subsystem 4 can facilitate the transfer of the sample back and forth between the thawing position and the initial position. The sample can be separated from the liquid nitrogen quickly at the thawing position. After separation, the sample recognition subsystem 3 is used for rapid identification. System 2 performs fast thawing. After thawing, the sample is quickly immersed in the thawing solution through the sample transmission subsystem 4, and the sample is transported back to the initial position after thawing; the existing manual control sample and laser pulse device are only an experimental device. To verify the feasibility of the laser thawing scheme in this embodiment, it is necessary to first align the unfrozen sample with a microscope at the laser irradiation position, and then press the sample into the liquid nitrogen and lock it through an ejection structure. While triggering the sample to eject liquid nitrogen to the set position, laser excitation completes the experiment. Since the sample will lose its activity quickly when exposed to the air, the frozen sample cannot be exposed to the air for a long time in actual use, and the user can use the microscope to align the laser irradiation position, so the device has no practical value. However, this device can apply the new technology of laser thawing in actual production, and can automatically complete the entire process from sample removal to thawing and recovery, which not only makes the process control accurate, but also improves work efficiency; at the same time, the system is effective for preservation. The freezing and thawing of difficult embryos and eggs has a very good effect, which is of great significance for the research and popularization of embryo and egg freezing methods.

Further, as a preferred solution, the sample transmission subsystem 4 of this embodiment includes a sample fixing device 41 and a sample transmission device 42, a liquid nitrogen transmission device 43, and a thawing liquid transmission device 44 that are respectively controlled by the main control module 1; The device 41 is used to fix the sample, the sample conveying device 42 is used to transport the sample from the initial position to the thawing position and return the sample to the initial position after thawing, and the liquid nitrogen transmission device 43 is used to quickly separate the control sample from the liquid nitrogen to facilitate The laser pulse generation subsystem 2 performs laser thawing, and the thawing liquid transmission device 44 is used to move the thawing liquid to the vicinity of the sample after the laser thawing, so that the sample is quickly immersed in the thawing liquid. As shown in Figure 2, the sample a is usually placed at the end of the front part of the rod b. The front part of the rod b is sleeved with a hollow rod cap c. The sample is located in the hollow structure of the rod cap c. When the rod b and the sample When a is frozen in liquid nitrogen, the liquid nitrogen will be filled in the hollow structure of the rod cap c. When the carrier rod b and sample a are taken out of the liquid nitrogen, the liquid nitrogen will temporarily remain in the hollow structure of the rod cap c. It still has a certain protective effect on sample a and will not rewarm immediately. When thawing is required, in the initial position, after the sample a is taken out of the liquid nitrogen tank, immediately fix the carrier rod b together with the sample a on the sample fixing device 41 (at this time, the rod cap c is also sleeved on the carrier rod b At the front, the liquid nitrogen still remains in the rod cap c), and it is automatically blocked; the sample a is transported from the initial position to the thawing position by the sample conveying device 42; the rod cap c is removed by the liquid nitrogen transmission device 43 to make the sample a is quickly separated from liquid nitrogen; the sample recognition subsystem 3 is used to quickly identify and locate the sample a. The laser pulse generation subsystem 2 laser thawed the sample a according to the preset parameters and trajectory, and the sample recognition subsystem 3 records simultaneously Laser thawing process; the thawing liquid transmission device 44 is used to move the thawing liquid to the vicinity of the sample a after the laser thawing so that the sample is quickly immersed in the thawing liquid; finally the sample conveying device 42 is used to transport the thawed sample a together with the thawing liquid The initial position is convenient for subsequent observation and analysis of thawing results.

In order to minimize the time the sample is exposed to the air, as a preferred solution, the liquid nitrogen transmission device 43 of this embodiment is provided with a liquid nitrogen box for containing liquid nitrogen, and a fixing rod for clamping is provided in the liquid nitrogen box. Cap clamping device. Therefore, when thawing is required, after the sample a is taken out of the liquid nitrogen box, the sample connecting rod cap c on the carrier rod b can be quickly immersed into the liquid nitrogen box in the liquid nitrogen transmission device 43 (liquid nitrogen box It contains liquid nitrogen), and the rod cap c is fixed by the clamping device, so as to ensure that the sample in the carrier rod b is completely immersed in the liquid nitrogen, and the sample will not be exposed due to the rapid volatilization of the liquid nitrogen retained in the rod cap c In the air; at the same time, the end of the rod b away from the rod cap c can be exposed to the liquid nitrogen box distribution, so that the liquid nitrogen transmission device 43 can be controlled to move closer to the initial position, so that the end of the rod b away from the rod cap c can be fixed on the sample On the device 41; the sample fixing device 41 and the liquid nitrogen transmission device 43 can simultaneously control the transport of the sample a from the initial position to the thawing position to ensure that the sample is always immersed in liquid nitrogen during the movement; while in the thawing position, when the liquid nitrogen After the transmission device 43 is removed with the liquid nitrogen box, the clamping device and the rod cap c, the sample a can be quickly separated from the liquid nitrogen. The ultimate goal is to minimize the time the sample is exposed to the air, ensure the activity of the sample to the greatest extent, and maximize the thawing effect of embryos and eggs.

In order to improve the accuracy of sample thawing, a laser energy control module and a laser pulse width control module are integrated in the main control module 1 of this embodiment. Before each laser thawing, the main control module 1 adjusts the laser energy parameters through the laser energy control module, and adjusts the laser pulse width parameters through the laser pulse width control module. Different biological samples (eggs or fertilized eggs) have different sizes and different energy required for thawing, which can be achieved by setting the laser energy and pulse width. Even if it is the same human egg, in the freezing process, due to the different amount of cryoprotectant used, the size of the frozen egg will also be different, and the energy required for thawing will also be different. Therefore, the main control module 1 can be used to set the energy and pulse width of each thawing, and cooperate with the sample recognition subsystem 3 according to the preset parameters to position the sample (egg or embryo), adjust the laser track, and emit the laser. Complete thawing.

To facilitate observation and analysis of the thawing results, the thawing system of this embodiment further includes a sample optical observation module 5, which is set in an initial position for optical magnification observation of the thawed sample.

To facilitate the user to control the entire system, the thawing system of this embodiment further includes a user interaction module 6 which is electrically connected to the main control module 1 for controlling the entire system through the main control module 1. Preferably, the user interaction module 6 of this embodiment includes a display, a touch screen, a mouse, and a keyboard electrically connected to the main control module 1 respectively. Therefore, the user can preset parameters of the main control module 1 through the touch screen, mouse, keyboard, etc., and realize the control of the entire system through the main control module 1; the thawing process can be easily observed through the display.

In order to optimize the structure of the system, the thawing system of this embodiment also includes a shared optical path system 7. The laser pulse generation subsystem 2 acts on the sample through the shared optical path system 7, and the sample recognition subsystem 3 performs sample identification and integration through the shared optical path system 7. Record the laser thawing process. The laser pulse generation subsystem 2 and the laser pulse generation subsystem 2 share the optical path system, so that the laser pulse hits the sample to unfreeze the sample, and the sample recognition subsystem 3 can simultaneously identify the sample and record the laser thawing process.

Furthermore, the thawing system of this embodiment further includes a power module 8 for supplying power to the main control module 1, the laser pulse generation subsystem 2, the sample recognition subsystem 3, and the sample transmission subsystem 4, respectively.

Industrial applicability

The above are only the preferred embodiments of the present invention, and do not limit the scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the description and drawings of the present invention, or directly or indirectly applied to other related The technical field is also included in the scope of patent protection of the present invention.

Claims

An automatic laser pulse thawing system for non-contact biological samples, which is characterized in that it includes a main control module, a laser pulse generation subsystem, a sample recognition subsystem, and a sample transmission subsystem;

The main control module is used to control the laser pulse generation subsystem, the sample recognition subsystem, and the sample transmission subsystem respectively, and make the laser pulse generation subsystem, the sample recognition subsystem, and the sample transmission subsystem work together;

The laser pulse generation subsystem is mainly used to generate laser pulses under the control of the main control module. The laser pulse generation subsystem includes a laser trajectory control module for controlling the trajectory of the laser pulse and a laser for controlling the emission of the laser pulse. A transmitting module, the laser trajectory control module and the laser transmitting module are respectively controlled by the main control module;

The sample recognition subsystem is mainly used to quickly identify, locate and record the laser thawing process of samples;

The sample transmission subsystem is mainly used to transport the sample to the thawing position, to control the sample to leave the liquid nitrogen quickly, to control the sample to be immersed in the thawing liquid, and to transport the sample back to the initial position after thawing.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, wherein the sample transmission subsystem includes a sample fixing device, a sample transmission device and a liquid nitrogen transmission device controlled by the main control module respectively. A device and a thawing fluid transmission device; the sample fixing device is used to fix the sample, the sample transfer device is used to transport the sample from the initial position to the thawing position and return the sample to the initial position after thawing, the liquid nitrogen transmission device It is used to control the rapid separation of the sample and the liquid nitrogen to facilitate the laser pulse generation subsystem to perform laser thawing, and the thawing liquid transmission device is used to move the thawing liquid to the vicinity of the sample after the laser thawing so that the sample is quickly immersed in the thawing liquid.
The laser pulse thawing system for automated non-contact biological samples according to claim 2, wherein the liquid nitrogen transmission device is provided with a liquid nitrogen box for containing liquid nitrogen, and the liquid nitrogen box is provided with There is a clamping device for clamping the fixed rod cap.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, wherein the main control module is integrated with a laser energy control module and a laser pulse width control module.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, wherein the main control module is integrated with a laser energy control module and a laser pulse width control module.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, characterized in that: it further comprises a user interaction module, and the user interaction module is electrically connected to the main control module for passing through the main control module. Realize the control of the entire system.
The laser pulse thawing system for automated non-contact biological samples according to claim 6, wherein the user interaction module includes a display, a touch screen, a mouse, and a keyboard that are electrically connected to the main control module.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, characterized in that: it further comprises a shared optical path system, and the laser pulse generating subsystem acts on the sample through the shared optical path system. The sample recognition subsystem performs sample recognition and records the laser thawing process through the shared optical path system.
The laser pulse thawing system for automated non-contact biological samples according to claim 1, characterized in that: it further comprises a power module, the power module is used to separately control the main control module, the laser pulse generation subsystem, the sample Identify the subsystem and the sample drive subsystem for power supply.