Research On Super-resolution Microscopy And Instrument Based On Multi-angle Illumination

The progress of biology and medical science has prompted developments of the optical microscopy,while the resolution enhancement is a popular topic.Due to the presence of diffraction limits,the resolution of far-field optical imaging is limited to about half a wavelength.In recent years,the super-resolution method based on fluorescence nonlinear technique has broken the diffraction limit to achieve the resolution of nanometre scale.However,these techniques require special dye labelling,slow imaging speed and strong phototoxicity,which greatly limits the application of these methods.Multi-angle illumination technology is a wide-field imaging method,which improves the resolution by spatially modulating the frequency spectrum of the sample.This method is suitable for scattering imaging,refractive index imaging and even fluorescence imaging.Multi-angle illumination super-resolution technology stands out as its fast imaging speed,slight sample damage and good signal to noise ratio(SNR),thus attracting more attention in the microscopy field.In this paper,according to the sample's characteristics,a multi-angle illumination microscopy system is designed to achieve super-resolution.The main work and innovation are as follows:(1)Traditional total internal reflection microscopy has the problem of nonuniform illumination.We propose a ring scanning TIRF imaging method,which not only realizes uniform illumination but also improves SNR.In the axial direction,we could achieve a resolution of 50nm by subdividing the illumination angle within the total reflection angle;In the lateral direction,the resolution is doubled by applying the ring scanning TIR method to the non-fluorescent sample imaging.(2)Traditional phase imaging technology is accompanied with low resolution.We propose a phase-only multi-angle optical field synthesis imaging method to enhance the resolution.This method applies the frequency extending theory of scattering medium in phase imaging,which can increase the effective numerical aperture(NA),improve the three-dimensional resolution of the phase image,and increase the SNR of the image.(3)The existing tomographic phase microscopy has the weakness of slow data acquisition speed and the poor controllability of the system.We propose a controllable tomography phase microscopy,which use piezoelectric ceramic to precisely control phase shift.We design a periodic phase shift algorithm that could select the scanning speed according to the characteristics of the sample.Our method's imaging speed is 4 times higher than that of the conventional method.(4)In order to avoid the poor phase resolution in large angle off-axis interferometry,we propose a tomography phase microscopy method using coaxial interference technology.This method guarantees the consistency of sample information obtained at different angles,and could increase the three-dimensional resolution of the refractive index by 20%.(5)Conventional structure illumination microscopy(SIM)is disturbed by the problem of artefacts.We propose a fluorescence imaging method combining the structure illumination technique and the frequency spectrum iterative algorithm.This method could obtain a resolution twice that of wide field fluorescence imaging,acquire much better SNR than conventional SIM,especially in the dense sample imaging.