High Speed Scanning Microscopy With Two-photon Fluorescence Of Mouse’s Bone Cells And CARS Of PMMA

In recent years,the two-photon and multiphoton fluorescence microscopy,coherent anti-Stokes Raman scattering(CARS)microscopic imaging,Second harmonic microscopic imaging and other imaging techniques based on non-linear optical effects all have made great progress,and become a current international research hotspot.Among them,CARS microscopic imaging has become an important way of chemical composition analysis and structural study of biological cells,tissues by its high resolution,high sensitivity,non-invasiveness,and availability of sample spectral information.Two-photon fluorescence microscopy with high resolution also play an important role in many fields such as biochemistry,pharmacology,and neurobiology due to its advantages such as deep imaging depth,low light damage,and multiple-label composite imaging.This thesis theoretically analyzes the above two types of nonlinear microscopy imaging and experimentally builds a set of femtosecond laser microscopy imaging systems that can simultaneously implement two-photon fluorescence microscopy imaging and CARS microscopy imaging.Two-photon fluorescence microscopy was performed on rhodamine-stained rat calvarial primary osteoblast skeleton samples and CARS microscopy was performed on PMMA particles to verify the advantages and applications of the two imaging methods in their respective fields.In terms of theory,the two-photon fluorescence generation process is first analyzed,the time average expression of the two-photon fluorescence intensity is deduced,and the locality advantage of two-photon excitation is verified theoretically,and the saturation state of the system is verified by derivation and simulation.In addition,in terms of CARS imaging,the expression of signal intensity in CARS is deduced and the method of suppressing non-resonant background noise is analyzed.Finally,the noise problems in the system are analyzed.In two-photon fluorescence imaging experiments,Rhodamine B samples were first used to confirm the two-photon microscopy imaging capability of the system.The prisms were used to realize pulse shaping of femtosecond lasers,and rhodaminestained rat calvarial primary osteoblast skeleton samples were microscopically imaged.Experiments show that the lateral resolution of this system can reach 1.52μm and the longitudinal resolution is 2.0μm.In the process of two-dimensional imaging,the effects of focal distance and pulse quality on the imaging quality were studied;the spatial structure and distribution of the microtubule skeleton in rat osteoblasts were observed during the tomographic and three-dimensional reconstruction process.The observations verify that the system has a vertical resolution of 2.0 μm.In CARS microscopic imaging experiments,microscopic imaging and background noise analysis of PMMA particles were performed.The comparison between the imaging results confirms the microscopy capabilities of CARS system and its micron-scale resolution.It was confirmed that the system constructed in this experiment can simultaneously perform two different nonlinear microscope imaging modes.