Biological Cells Microsurgery Induced By Femtosecond Pulsed Laser

Femtosecond laser is an ideal tool for three-dimensional micro and nano fabrication in any transparent materials because of its ultrahigh temporal and spatial resolutions and peak power. This technique will improve the development of information technology, biological technology and MEMS. Firstly, femtosecond laser micro/nano fabrication experiment system is established in this thesis. Then, surface modification of solid materials and the nucleus micro/nano surgery in vivo are achieved using this system. Lastly, we studied on the femtosecond laser protuberance surgery of olfactory ensheathing cell and flagellum surgery of Chlamydomonas. This work has important significance for many scientific domains, such as micro and nano fabrication and single cell scientific research.Firstly, we established femtosecond laser micro/nano fabrication experiment system, which integrate ultrafast laser technology, microscopy technology, three-dimensional scanning technology and computer controlling technology. This system is composed of Integra-C femtosecond laser, light transmission system, and super-smart orientation system. Then, we introduce work principle and process of femtosecond laser micro/nano fabrication experiment system.On the glass surface, the micro-grooves are fabricated with Femtosecond laser experiment system. The influence of experiment parameters on the fabrication are not only analyzed theoretically but also validated experimentally. The olfactory ensheathing cells are cultured on the smooth glass surface and micro-grooves surface, and we found the microgrooves affect the orientation of cells. The Hella nucleoluses, whose histone are demarcated by green fluorescent protein, are incised in this experimental system and achieve a resolution of about 300nm. Subsequently, we study the cell activity after micro-damage in the nucleus.The protuberances of the olfactory ensheathing cell are cut by femtosecond laser surgery system, and the influences of laser power and scanning speed on the surgery effects are studied firstly. The femtosecond laser surgery on cellular protuberance can induce intracellular waves in a living olfactory ensheathing cell. The calcium wave generation mechanisms are further investigated using different culture mediums and protuberance diameters. Based on these results, we conclude that shockwave-induced mechanical force and laser-induced cytoskeleton depolymerization are two key factors. Furthermore, the cell activity after protuberance surgery is observed and the role of calcium wave in this process is analyzed.Lastly, we use tightly focused femtosecond laser to radiate Chlamydomonas flagellum. Under certain laser power range, the flagellum repture in its root and the reason for this phenomenon is discussed initially. After surgery, the Chlamydomonas remain its activity and the regeneration of flagellum is observed.