Femtosecond Fiber Laser For Deep Biological Tissue Multiphoton Microscopy

In recent years,based on the rapid development of ultrashort pulse laser techniques,multiphoton microscopy has been widely been concerned and successfully applied to the fields of biology,medical science,nanomaterials sciences and the basic research and so on.Multiphoton microscopy has many advantages,such as deep imaging depth,small light damage,high spatial resolution and high fluorescence collection rate.The key factor to affect the imaging performance of the system is the laser source.Currently,there are many kinds of laser sources applied in multiphoton microscopy system,among which the ultrashort pulse fiber laser is more prominent.Due to its advantages of compact,low price and excellent performance,it is of great significance to the frontier application and product transformation of multiphoton microscopy.At the same time,the application of multiphoton microscopy also puts forward higher requirements for the ultrashort pulse fiber laser.In the process of imaging biological tissue,it is required to develop a laser source that can be used to image deep biological tissue.Therefore,this dissertation focuses on development ultrashort fiber lasers for deep tissue multiphoton microscopy including erbium-doped and ytterbium-doped femtosecond fiber laser,and 1.7 ?m femtosecond fiber laser.The preparation and staining of cell tissue and other biological samples were studied.The main work and innovations of this dissertation are summarized as follows:1.We realized an all-fiber erbium-doped femtosecond fiber laser is studied by different mode-locking mechanisms.First,all-fiber erbium-doped femtosecond laser based on carbon nanotube saturated absorber is realized with central wavelength of 1560 nm,spectral width of 5.5 nm,repetition rate of 33.5 MHz,average power of 10 mW,and pulse width of 550 fs.In order to improve the stability of the laser,we realized all polarization-maintaining femtosecond fiber laser,based on nonlinear amplifying ring mirror,with central wavelength of 1560 nm,spectral width of 18 nm,repetition rate of 102 MHz,average power of 64 mW,and pulse width of 180 fs.And then,the power is amplified to 285 mW by the all-fiber amplifier.The pulses width is ?60 fs.The system is designed as a full polarization-maintaining fiber,with excellent mode-locking start-up characteristics,good stability and excellent environmental stability,so it is suitable to various applications.At the same time,it is applied to the study of fiber frequency comb.2.By using the soliton self-phase frequency shift effect of fiber,all polarization-maintaining 1.7 ?m fiber laser is realized.First,we theoretically analyzed soliton self-frequency shift brought by the pump laser and optical fiber parameters.And then,based on erbium-doped femtosecond fiber laser,all polarization-maintaining 1.7 ?m femtosecond laser has been achieved by using 460-m polarization-maintaining fiber.The average power is 35 mW,the center wavelength is 1.7 ?m,the pulse width is 368 fs and light conversion efficiency is 66%.This system is all-fiber design,with compact structure and stable performance.3.Ytterbium-doped femtosecond fiber laser was obtained by using nonlinear polarization rotating mechanism and the phenomenon of dissipative soliton bound state was observed for the first time.Firstly,we realized all-normal-dispersion ytterbium-doped femtosecond fiber laser.The central wavelength is 1047 nm,the spectral bandwidth is 15 nm,and the pulse width is 10.7 ps.After being compressed by the external cavity grating,the pulse width is 200 fs.At the same time,bound state of dissipative soliton was observed experimentally for the first time.In order to further obtain shorter pulse,femtosecond pulse laser is obtained by dispersion management.The average power is 80 mW,the spectral bandwidth is 50 nm,and the repetition rate is 50 MHz.The pulse width is 56 fs after being compressed by the external cavity grating.With stable performance and compact structure,the 1.0 ?m femtosecond laser can be used as a seed source for the subsequent 1.3 ?m femtosecond laser.It provides the technical support for deep tissue multiphoton microscopy system.4.Biological samples were prepared and imaging studies were carried out by fluorescence microscope.Firstly,rat brain slices were prepared,the nerve cells were stained with Nissl staining,and the morphology of the cells was imaged.Secondly,the primary neurons of rats were obtained by surgery,the neurons were stained with a fluorescent dye called ghost ring peptide,and the structure of the nerve cell cytoskeleton was imaged.In addition,the culture of fibroblast living cells was completed,and the dynamic changes of the cells under inflammatory conditions were tracked by fluorescence imaging.We observed NF-?B entering and leaving the nucleus after TNF-? stimulation.This work will make necessary preparations for the imaging capability test and application of deep tissue multiphoton microscopy system.