【作者】 巩继贤；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2007， 博士

【摘要】 单细胞技术在研究细胞的生化组成与行为特性的关系,细胞之间及细胞与环境的关系和胞内反应动力学等方面有重要意义,对于高产株的筛选等生物工程问题的解决也有方法学的意义。飞秒激光的高时间分辨率和空间分辨率以及以极小的单脉冲能量便可获得极高的峰值功率的特性,使其在单细胞研究中有独到的优势。本文以雨生红球藻和红发夫酵母为研究对象,以飞秒激光显微操作系统为实验平台,围绕在单细胞水平的虾青素高产株选育,进行了系列研究。研究了飞秒激光光镊对活体单细胞的捕获。测出了飞秒光镊捕捉细胞的Q值,比较了飞秒激光光镊和连续激光光镊对单细胞的捕获能力,从理论和实验两个方面证明了飞秒激光和连续激光一样可以作为光镊的光源使用。另外,对光子晶体光纤用作光镊进行了初步的尝试。首次进行了飞秒光镊对细胞活性影响的系统研究,通过研究飞秒光镊的功率和作用时间对细胞活性的影响,给出用飞秒光镊进行活体单细胞操作的参数范围。以飞秒光镊对单细胞的捕捉为基础,成功实现了细胞悬液中活体单细胞的转移培养。首次将飞秒激光作为诱变方法用于虾青素高产株的选育。分别对雨生红球藻和红发夫酵母用飞秒激光在单细胞水平进行了诱变处理,结果表明飞秒激光对雨生红球藻的单细胞诱变有一定的效果。在用光谱的方法直接检测单细胞的虾青素含量方面做了初步的探索,实现了红发夫酵母单细胞中虾青素含量的测试。首次实现了飞秒激光诱导细胞融合。以飞秒激光显微操作系统为实验平台,以红发夫酵母为对象,实现了飞秒激光诱导的细胞融合;对影响飞秒激光诱导细胞融合的因素进行了分析;在此基础上,对其机理进行了研究,提出了飞秒激光诱导细胞融合的模型。更多还原

【Abstract】 Single-cell techniques have been used to describe the composition and activities of cells, have been a key element in revealing the relationship of environmental distribution and activities of microorganisms, and have been used to detail dynamics of reactions in cells. Single-cell methods have also been essential to understanding problems in bioengineering such as the screening of high-product strains. Femtosecond laser has attractive in single-cell research for high temporal resolution and high spatial resolution. Study of astaxanthin biosynthesize at single cell level was performed with Haematococcus pluvialis and Phaffia rhodozyma by femtosecond laser manipulation in this article.The trapping of living cells by femtosecond laser tweezers was been studied. Q-value of femtosecond laser tweezers was measured and the trapping capability of femtosecond laser and continuous wave was compared. The result proved that femtosecond laser could be used as optical tweezers as well as continuous laser. Additional, a primary attempt was made in using optical crystal fiber as optical tweezers.A system study of activity of cells in the trapping of femtosecond laser tweezers was performed by the analysis of the effect of laser power and exposure time on cell activity. The range of laser power and exposure time for safe trapping has been provided. The transfer of single cell from the suspension to plate has been achieved successfully on the base of the trapping by femtosecond laser.Femtosecond laser pulse has been used as the method of mutagenesis for the first time. The cells of Haematococcus pluvialis and Phaffia rhodozyma have been treated with femtosecond laser at single cell level. The results showed that the mutagenesis by femtosecond laser at single cell level succeed for Haematococcus pluvialis while failed for Phaffia rhodozyma. A primary attempt was carry out in detecting of astaxanthin content in single cell by microspectroscopic method.The preparation of protoplast of Haematococcus pluvialis and Phaffia rhodozyma has been accomplished through the selection of appropriate enzyme and the analysis of conditions.Femtosecond laser induced cell fusion was achieved for the first time. The fusion of Phaffia rhodozyma protoplast was performed with femtosecond laser manipulation system. The factor which may affect the result of femtosecond laser induce fusion was investigated and the possible mechanism was studied. A hypothetical model of cell fusion induced by femtosecond laser was presented on the base of experimental result.更多还原