Mapping The Dipoles Orientation Distribution With Super Resolution Via Fluorescence Polarization Modulation

Fluorescence polarization microscopy(FPM)is widely used to obtain the orientation and structure information of biomolecules.Geometric models are used in all related methods to statistically describe the orientation distribution information of biomolecules labeled by fluorescent molecules in the excitation spot,the resolution of which depends on the spatial resolution of the microscopy imaging system.Therefore,improving the spatial resolution of the fluorescence microscopy system is the key to obtain more accurate molecular orientation distribution and finer molecular structure information.Recently,researchers have proposed fluorescence polarization-modulated super-resolution technique.This technique has the same polarization excitation scheme with FPM,and is naturally suitable for the research of super-resolution biomolecular orientation distribution imaging in principle.However,the super-resolution reconstruction algorithm proposed by this technique requires the sparsity constrained regularization terms,the influence of the corresponding coefficients on the reconstruction process and results limits its application.In view of the above problems,this paper has carried out researches on super-resolution imaging and super-resolution molecular orientation distribution imaging based on fluorescence polarization modulation.The main contents are as follows:Firstly,based on the fluorescence polarization modulation microscopy imaging theory,an optimization model is established to minimize the residuals,and a new super-resolution microscopy imaging reconstruction algorithm is developed based on the conjugate gradient method.The contribution of polarization modulation to super-resolution imaging is proved by comparing the wide-field image,the deconvolution image and the reconstruction image of the sample.At the same time,the effectiveness of this algorithm is proved via the analysis and comparison of the microscopic image reconstruction results of biological samples.On this basis,a super-resolved molecular orientation distribution imaging method via fluorescence polarization modulation is further proposed.In this method,a geometric model describing the orientation distribution of dipole clusters is established,and then according to this model and polarization modulation microscopy imaging theory,the previous reconstruction algorithm is improved and the SR-OM(Super Resolution-Orientation Mapping)joint reconstruction algorithm is developed.This algorithm does not need to introduce any regularization terms,and is able to reconstruct multiple significant parameters to simultaneously acquire the super-resolution image and effective orientation distribution information of dipole-clusters at corresponding resolution.The reconstructed results of different samples prove that this method can obtain the spatial resolution of quarter wavelength and the orientation distribution image of fluorescent dipole cluster at the corresponding resolution.Furthermore,based on the Monte Carlo method,the accuracy of reconstructing the molecular orientation distribution information of this method was studied and analyzed.