Numerical And Experimental Investigations On Spatial Light Modulation And Aberration Compensation In Microscopy System

In recent years,with the development of big data storage,laser processing,optical imaging,optical encryption,optical communication and optical tweezers,laser beam modulation has been widely used.Light field modulation based on spatial light modulator is an effective way to realize different shape and number of focal spots,which have been widely concerned by researchers in recent years.For example,in laser processing,the complex spot pattern obtained by beam modulation provides the possibility for the processing of complex micro/nano-structure.In the field of microscopic imaging,multi-beam modulation can effectively improve the imaging speed,which is very important for the imaging of samples,especially the biological samples sensitive to phototoxicity and easy to bleach.At the same time,in microscopic imaging,since the beam is focused on the biological sample through the objective lens,the widely existed difference of interface refractive index will cause aberrations,which will reduce the resolution of the microscope and lead to measurement errors.This paper studies theoretically and experimentally:(1)the method of generating multifocal spots in a high numerical aperture microscope system based on the spatial light modulator,and(2)the influence of tilted interface with mismatched refractive indices on fluorescence microscopy imaging,and proposes the corresponding phase compensation method.The main research results are summarized as follows:1.Based on Debye diffraction integral theory,a strip segmentation phase method is proposed to generate uniform and controllable multifocal spots on the focal plane of the objective lens of a high NA micro-imaging system through a spatial light modulator.The strip segmentation phase method can be combined with blazed grating to remove the influence of zeroth-order light.The results of numerical simulation and fluorescence imaging show that the uniformity of multifocal spots is more than 99%.Finally,by dynamically loading the phase diagram of the SLM and combining it with the single-photon laser direct writing technique,the multifocal spots parallel laser processing of different structures is realized on the high numerical aperture microscope system,and the fabrication accuracy reaches 0.875 μm.2.In order to achieve more flexible and diverse spot modulation,based on the strip phase segmentation method,fluorescence spots of the perfect vortex and chiral light spot have been realized.In addition,by combing the strip segmentation phase,vortex phase and axicon phase for segmentation,the perfect vortex light and Bessel light are generated on the focal plane of the objective lens simultaneously.This modulation may provide a more flexible way for laser processing of complex bionic structures.3.The influence of tilted interface with mismatched refractive indices on microscopic imaging is studied.Firstly,based on the diffraction integral theory,the aberration of the tilted interface with mismatched refractive index in a laser scanning microscope and its influence on the focal spot of the objective lens are studied theoretically.It is found that the focal position,shape and FWHM are significantly affected by the tilted interface with mismatched refractive index,including:(1)the focal position is significantly shifted along the beam axis,which will lead to positioning errors such as confocal and stimulated emission depletion microscopy in three-dimensional(3D)target reconstruction;(2)the FWHM of the focal spot is increased and the spatial resolution of the system is reduced.In order to correct the effect of the tilted interface with mismatched refractive index,we propose a phase-only modulation method.By loading the phase compensation map into the spatial light modulator(SLM),the effect of the tilted interface with mismatched refractive index is corrected.This study provides an effective method for spatial light modulation of complex light field,and an effective way for the researches and applications of fast optical imaging,laser processing,optical communication and optical tweezers.At the same time,the significant influence of the tilted interface with mismatched refractive indices on the imaging system of laser scanning microscope is revealed.The correction method is proposed,which is of great value to achieve accurate biological structure imaging research.