Study On Super-resolution Method Based On Coherent Anti-stokes Raman Scattering

After decades of development,there have been quite a few methods for super-resolution microscopic imaging technologies.However,in the field of biological microscopic imaging,mainstream super-resolution methods(such as electron microscopy,near-field microscopy,or fluorescence microscopy imaging methods)have obvious limitations,and are difficult to meet all requirements at the same time,such as high resolution,label-free,no toxicity and no damage.As a non-linear method for detecting the vibration state of molecules,Coherent anti-Stokes Raman scattering(CARS)technology can simultaneously obtain information of multiple substances in focus without introducing exogenous labels to samples.Therefore,CARS is a proper detection method for living samples.In recent years,many kinds of super-resolved CARS microscopies were put forward,but seldom verified experimentally.In this paper,we have made some explorations on super-resolution microscopic imaging methods based on CARS.First,We have carried out relevant experimental verification of additional probe-beam-induced phonon depletion(APIPD)theory,including phonon depletion experiments and competition experiments.Then according to the experimental results,we discussed the feasibility of this method.Finally,the article first proposed the axial superresolution technology of CARS based on the modification of the double helix function.This method can well improve the axial resolution.And on this basis,simulation and optical system design and other related work were carried out.The specific research work is as follows:1.In order to verify the feasibility of APIPD,we first built a three-color CARS detection system.The system uses a fiber laser with a center wavelength of 1064 nm to pump a photonic crystal fiber and a nonlinear crystal as light sources.Then an automatically controlling software was completed by Lab View.Finally,combined with the method of time delay,the backgroundfree CARS signal of the benzonitrile solution and the relationship between the CARS signal and the power of the probe light are obtained.2.In theory,two probe lights with different center wavelengths will produce different CARS signals.However,due to the time sequence,there will be signal competition and loss.Therefore,based on the three-color CARS system,we designed and added an optical path for generating additional detection light,and built a four-color CARS system.The PPLN crystal was used in the system to generate pulses with a center wavelength of 808 nm which was used as the additional probe.And use the fully automatic Labview control program to control the pulse timing.Finally,a series of confirmatory experiments were conducted to discuss the possibility of APIPD theory.At the end,the APIPD theory was discussed according to the phenomena in the experiment.3.An axial super-resolution CARS approach based on double helix point spread function(named CARS-DH)is proposed.And completed the preliminary simulation work,including phase plate design,single molecule CARS-DH signal,and z-axis calibration.In addition,two CARS-DH-based system designs are proposed in this paper.They are the broadband CARS signal detection optical path based on the frequency scanning of the optical parametric oscillator and the detection optical path using fiber bundles as detectors.The latter can accomplish the three purposes of broadband spectral measurement,lateral and axial high resolution,and highspeed detection simultaneously.The main innovation in this thesis can be summarized as follows:1.On the basis of the proposed APIPD theory,a three-color and four-color CARS competition system is built.Confirmatory tests related to signal saturation,signal competition and pseudocompetition have been conducted successively.Then we discussed the series of data obtained.2.A CARS microscopy with axial super-resolution was first put forward,which is based on point spread function engineering.This method can keep all advantages of CARS itself,and furthermore,it can provide axial super-resolution information of samples.According to this method,preliminary simulation work was carried out,and two microscopic imaging systems based on CARS-DH.