Research On Sampling Method Of Tissue Section Micro-Area

In recent years,technologies such as single-cell sequencing and spatial transcriptome sequencing have developed rapidly due to their ability to dig deeper into cell heterogeneity information.Single-cell omics can obtain single-cell resolution information.The investigation of cell heterogeneity is helpful for cell classification,time series analysis,tumor analysis and other research,which can solve the problem of untrue information encountered in the traditional analysis of multiple cell sample research and using average data instead of single cell research.The application of single-cell transcriptome sequencing technology has greatly broadened people’s horizons,enabling people to deeply understand the diversity of cell composition and gene expression status in tissues.Single-cell transcriptome sequencing technology can be used to analyze the changes in cell types and gene expression in the time dimension.However,gene expression is also spatially specific.Traditional single cell sequencing lost spatial information,but spatial transcriptome sequencing can retain the spatial location information of cells and reveal the interactions between cells.Single cell isolation or tissue micro-area separation is the first step of single cell sequencing and spatial transcriptome sequencing.The quality of sampling directly determines the results of subsequent analysis.In this paper,we explored micro-sampling methods for tissue sections based on microneedle and laser-induced forward transfer(LIFT)sampling.Microneedles sampling is a sampling method based on traditional mechanical micromanipulation,and LIFT is a new technology based on pulsed laser.The main research contents of this paper are as follows:(1)Study on sampling method based on microneedle: The micromanipulation device is equipped with capillary microneedles,the micro-sampling of tissue sections is observed in real time under the microscope,and the micro-sampling of tissue sections is carried out.The sampling effect and the influence of various parameters on the sampling are investigated.Using PDMS slides for microneedle sampling is beneficial to protect the integrity of microneedle and improve the success rate of sampling.The smaller the size of the microneedle tip,the higher the sampling quality and the higher the success rate.The sampling integrity and sampling efficiency are improved by using capillary microneedle sampling in the controlled gas path of micropump.Microneedle sampling can achieve single-cell size of tissue sampling and the sampling efficiency can be up to 80%.(2)Study on sampling method based on femtosecond LIFT: First,the LIFT device was designed and built.The optical path was designed based on the femtosecond laser,the presampling preparation was completed.Then,the sampling feasibility was verified through the experimental processes of sampling,collection,PCR and gel electrophoresis.In addition,the influence of aperture size and laser power on the sample size is explored,and the experimental results show that the sample size increases with the increase of aperture and laser power.(3)Development of picosecond LIFT sampling instrument: The impacts of nanosecond,picosecond and femtosecond lasers on LIFT have been discussed.The easy-to-integrate miniature picosecond fiber laser was selected as the laser light source of LIFT,the LIFT instrument was assembled,the feasibility of picosecond fiber laser for LIFT sampling was studied.And the influence of laser current on the sample size is investigated.In addition,the advantages and disadvantages of LIFT and laser capture microdissection(LCM)are compared,and the experimental results prove the practicability of the LIFT sampling instrument developed in this paper.It highlights the advantages of LIFT instrument such as low-damage and high efficiency.