Development Of Cryogenic Microscopic Imaging System Based On Instant Contact Method And Freeze-thaw Visualization Experiment

Infertility has become the third major factor affecting human health after tumors and cardiovascular and cerebrovascular diseases.More and more patients are using assisted reproduction technology of gamete freezing to solve the problem of infertility,and cryopreservation of cells is One of the key links.Thanks to the development of low-temperature microscopic imaging technology,people can observe the entire process of freezing and thawing of cells and cryoprotectants,thereby studying the mechanism of cell freezing and thawing and improving the success rate of freezing.At present,cryogenic microscopy imaging systems mostly use liquid nitrogen as the cold source,and the samples are frozen by reducing the temperature of the freezing platform through heat conduction or convection.However,due to the influence of system leakage and other problems,the highest cooling rate that can be achieved is mostly About 150 ℃/min,while the cooling rate of cells during cryopreservation can often reach 2000 ℃/min,the system is far from reaching the cooling conditions for cells during cryopreservation;in addition,in traditional cryogenic biological microscopes,the sample is directly exposed to the open environment.The cold stage is exposed to heat exchange,but the gas atmosphere and gas pressure of the environment where the cells are located will also have a certain impact on the cryopreservation of cells.Therefore,in response to the research needs of cryopreservation of cells,a new type of cryogenic microscopic imaging experimental system was developed,and the visual experiment of liquid freezing and thawing was carried out with this system.The specific research content of this article includes:(1)A new freezing method based on the instant contact method is proposed.The concept of an environmental chamber is introduced into the low-temperature visualization system.The principle of excellent thermal insulation performance between objects that do not touch each other in a vacuum is used.High temperature difference and sudden increase in thermal conductivity to achieve rapid cooling.The specific implementation plan is to place the biological sample in an independent small environmental chamber,the internal gas atmosphere is controllable,the outside is insulated by a vacuum chamber,and a liquid nitrogen heat sink is also installed in the vacuum chamber.By controlling the environmental chamber and the liquid nitrogen heat sink The contact or separation corresponds to rapid cooling and rewarming respectively.At the same time,a numerical model of the core heat exchange area of the system was established,and the influence of the size and thermophysical parameters of the components in the environmental cabin on the cooling rate and temperature distribution of the stage was analyzed,and the specific design of the environmental cabin was determined based on the above numerical simulation results.(2)The rapid freezing scheme based on the instant contact method built an experimental system for low-temperature microscopy imaging,designed microscopic imaging,temperature measurement and pressure measurement and control systems for the new freezing scheme,and debugged the system’s cooling and reheating capabilities.In order to further study the cooling performance of the experimental system,the influence of the interface contact thermal resistance on the cooling performance was analyzed through experimental measurement and the experimental system was optimized based on the measurement results.The optimized system has a cooling performance of up to 1600 in the temperature range of 300-250 K.℃/min;(3)Using pure water and cryoprotectant as the research object,the visual experiment of liquid freezing and thawing was carried out using this new lowtemperature microscopy imaging system,and the pure water and cryoprotectant were photographed in droplets,circular tubes,and thin layers.Under the freezing and thawing process under different stage temperature conditions,combined with the ice crystal growth theory,the speed and morphological characteristics of ice crystal growth under different subcooling degrees are analyzed.Preliminary cell freezing and thawing experiments were carried out.Through the comparative study of ice crystal growth characteristics and the freezing and thawing experiments of cells in cryoprotectant solution,the protective effect of cryoprotectants on cell freezing and thawing was analyzed.Through the research of the above three parts,the principle of instant contact rapid freezing is explained,the experimental system is optimized through numerical simulation and experimental measurement,and the experimental system built is used to study the pure water and cryoprotectant under different forms and different degrees of supercooling.The freezing and thawing process of the cell,to a certain extent,reveals its freezing and thawing characteristics,and lays the foundation for the future study of cell freezing and thawing mechanism.