Multi-color Multi-region Multiphoton Excitation Fluorescence Microscopy

Fluorescence test technology is one of the most important method to biomedical.,had been widely used in dairy life. Fluorescence inherently is a multi-parameter signal, Fluorescence measurement can be used to identify different fluorophores,Fluorescence lifetime had proved to identify various different overlapping sample with high resolution. But the traditional two photon fluorescence microscopy imaging technique can not get the fluorescence information of the complex regions of the complex samples fast. If just use a laser as the light source of the whole system to carry out the whole excitation imaging, region of interest outside of the samples produced photon damage, reduce the rate of energy utilization, and the time resolution is poor..In this paper, we propose multicolor multiphoton excitation fluorescence ima--ging technique based on supercontinuum light source and multiregion multiphoton excitation fluorescence microscopic imaging technology based on temporal focusing. Multicolor multiphoton technique based on supercontinuum light source of containing multiple fluorophores samples to achieve the most optimum excitation efficiency of fluorophores quickly.We also propose a multiple regions multiphoton fluorescence microscopy based on temporal focal technology. Can aim at several different intere--sting regions meanwhile generating different arbitrarily matching spot due to phase shaping function provided by spatial light modulator and temporal focal based on flash grating. Finally utilize linear array CCD to collect signal. This technology allow for aiming at multiple regions of interest addressing imaging under the condition of no damage to other parts of biological samples also with high spatial and temporal resolution.In summary, this thesis focus following issues:1,Introduced advantages and disadvantages and development status of Two-photon fluorescence microscope imaging.2,The mechanism of two-photon fluorescence、SLM and Temporal focal.3,Multicolor two-photon excitation fluorescence microscopy imaging technology research. It mainly introduces the build process of super-continuous spectrum light source and two-photon fluorescence microscopy imaging technology based on super-continuum spectrum light source, finally utilize Leica rhizome sample to achieve multicolor multiphoton excitation fluorescence imaging based on system we proposed.4,Regional multiphoton microscopy imaging research, mainly introduces the design、construction and resolution calibration of the multi regionals addressing temporal focal multiphoton fluorescence microscopy system. Finally get imaging of Hella cells, pollen cells and other biological samples labeling by rhodamine based on system we propose. The experimental system has broad application prospects.