Research On Ejecting Isolation Of Single Microbial Cell Based On Laser-induced Forward Transfer

Single cell study can provide a deeper understanding of life activities,and in which isolation of single cell is basic.Commonly used cell sorting methods include limiting dilution,micromanipulation,flow cytometry,laser microdissection,microfluidic cell sorting,optical tweezers,and magnetic sorting,and each method has its advantages and disadvantages.Due to the ability to transfer tiny substances,Laser-induced forward transfer(LIFT)is widely used in industrial and scientific fields such as printing electronics,microfabricating 3D structures,and bioprinting of cells.The application of LIFT to eject microbial single cells currently has the following problems: First,the receiving ratio of single microbial cell is low.Due to the inappropriate laser energy,the ejected cell's structure is not complete,or the ejected cell cannot be successfully received.Second,live ejection and cultivation of single microbial cell cannot be achieved.The heat,force and other factors in the ejection process could cause damage to the cells,so that the ejected cells cannot be cultured;Third,the range of ejected microorganisms is limited.Currently,the size of microorganisms that could be ejected is 0.5-20?m,and microorganisms with larger sizes and complex shapes cannot be ejected.For the above problems,this thesis mainly focuses on the interaction between ns-pulsed laser and aluminum film,and carried out the research on LIFT for single microbial cell ejecting isolation.The research content and innovative achievements are as follows:(1)We designed and built a single-cell ejection sorting system based on laserinduced forward transfer,and compared different materials on microbial single-cell ejecting and receiving under continuous laser.Aiming to improve the receiving rate of the ejected microbial cells,we irradiated aluminum film with small energy ns-pulsed laser to eject microbial cell by the thermal deformation of the film,and the receiving efficiency of ejected microorganisms is 97%.Finite element simulation is carried out to calculate the temperature distribution and thermal deformation of the aluminum film.And laser scanning confocal,SEM,AFM,high-speed imaging methods were utilized for characterization.(2)For live ejection and culture of single cell have not been achieved,we designed a thermal insulated structure,to prevent the thermal damage that the high-temperature aluminum film brought to microbial cell in the ejection process,and to eject the cells by the deformation of the insulation material.The temperature field is calculated using finite element method simulation,and the results show that when the temperature of the aluminum film rises to the ejection threshold,the microbial cell has already been ejected,so the heat didn't cause damage to the microbial cell because of the thermal insulation.Through experiments,single-cell culture rates of 63%,74%,and 22% were achieved for Yeast,Escherichia coli,and Lactobacillus,which verified the effectiveness of the thermal insulation structure.Further,combined with fluorescence imaging,target cells with fluorescence were sorted from mixture samples and complicated soil samples,and achieve a single-cell culture rate of 13.6% in complex soil samples.(3)Aiming at solving the problem that single ejecting spot cannot eject microorganisms with complex shape and large size(greater than 20?m),we combined beam shaping and laser-induced forward transfer technology.The laser beam is modulated into multiple spots,which can successfully eject Lactobacillus,Anabaena,and Flagellates with complex shapes and a length of 100?m,also eject 7 single yeast cells simultaneously.These results verified the feasibility of this technology and expanded the application of LIFT for ejecting and sorting of microbial cells.