Study On Parametric Technique And Broadband Four Wave Mixing CARS Microscopy

Coherent anti-Stokes Raman Scattering（CARS）microscopy shows the advantage of non-invasive sample detection due to the use of molecular-origin chemical bonds,good lateral resolution and three-dimensional chromatography for the third-order nonlinear effect itself,which has attracted more and more research interests from different areas.However,up to date,how to fast gain the steady lateral resolution of broadband CARS imaging system remains to be a challenge.For conventional single-frequency CARS systems,polystyrene microspheres are frequently used to measure the resolution of the CARS imaging system by imaging specific chemical bonds,but the excitation energy is required to disperse to all chemical bonds.The excitation power of single-frequency CARS is markedly reduced,compared to the broadband CARS.In the meanwhile,the polystyrene samples are supposed to possess high excitation power tolerance,which can endure long exposure time and relatively high power of excitation laser to maintain its original form.In general,larger samples in size can endure a little higher laser power,and the image of the measurement system is equal to the convolution between the sample size and the point spread function of imaging system,such as all fluorescence imagings.However,in CARS imaging,when sample size is larger than the system point spread function,the convolution relationship cannot work well.Two-photon fluorescence and CARS belong to the third-order nonlinear effect,and the theoretical formula shows same core of the resolution‘s theoretical value.When the excitation wavelength is identical,the resolution is mainly denominated by the order of the non-linear effect.The two-photon fluorescence resolution may therefore represent the resolution of the CARS system.Rare two-photon fluorescent materials can work well under very short excitation wavelength.In addition,because of high power and long exposure time of broadband CARS,the resolution of the broadband CARS spectral imaging system should be obtained quickly and the stability of this process should be maintained.Therefore,the deeper nonlinear four-wave mixing mechanism of the CARS process must be studied to find a new way to obtain a stable,fast spatial resolution in a broadband CARS spectral imaging system.Research demonstrated that the lateral spatial resolution of the system was obtained by deconvoluting the two-photon fluorescence image and the nonlinear four-wave mixed image with a small material of high power endurability.The main contents of this dissertation are itemed as follows:1.Using the semi-classical theory to analyze the CARS process and also to deduce the general four-wave mixing process,meanwhile to do in-depth analysis on the energy level of the nonlinear four-wave mixed signal generation mechanism,and then compare the relationship between the CARS resonant signal and the nonlinear four-wave mixed signal.2.Using the formulas of the two-photon fluorescence and CARS signals to examine numerical fitting and do a literature comparison to find the cause of the difference between the two nonlinear effects.3.A FDTD numerical analysis of the CARS theory is done to find the cause of the difference between the images.A spectral analysis method based on the maximum entropy method is proposed to deal with the broadband CARS image.4.The relative displacement between the axis of the beam and the center of the glass bead is used to analyze the internal mechanism of the parametric process to determine why the lateral image size is smaller than the actual sample size.5.Based on our own developed broadband CARS imaging instrument,spectral analysis with polystyrene fluorescence beads and pure polystyrene beads is done to compare two-photon fluorescence and broadband CARS spectral imaging.SiO₂ beads are also used to measure the lateral spatial resolution of the CARS imaging system based on the nonlinear four-wave mixing signal.The main innovations involved in this dissertation:1.Numerically analyzing the difference between two-photon fluorescence and CARS imaging and comparing the results with the literature.2.Using the self-built dynamic model to do numerical analysis using relatively large quartz beads when the center of the spherical and optical axis has a certain displacement.This results in a non-linear four-wave mixing image of a lateral size that is smaller than the actual sample size.The results of theoretical analysis and numerical analysis show that the momentum conservation and energy conservation conditions should be satisfied for the four-wave mixing at the same time.3.Using the semi-classical theory analysis of the four-wave mixed signal generation mechanism and determining its characteristics using the parametric process perspective.The resolution of the CARS system is measured by the four-wave mixed non-resonance signal.The theoretical analysis and experimental results are consistently very good.This broadens the awareness of four-wave mixing,which reduces the complexity of the system by selecting the appropriate high temperature resistant material and saving time while also increasing the repeatability and stability of the test.