Study On The Imaging Quality Of STED-4Pi Microscopy

With the development of new fluorescent molecular probes and different imaging experimental methods,a variety of new super resolution fluorescence microscopy techniques appeared.They are several or even more than ten times higher resolution than traditional techniques.However,due to system aberration,the imaging quality of super resolution microscopy is affected.Therefore,this paper mainly discusses the effect on imaging quality of STED-4Pi super resolution microscopy caused by aberrations.This paper also provides the basic theory about the compensation and optimization of system aberrations,and how to denoise the image generated by STED-4Pi super resolution microscopy for improving the system imaging quality.Base on the size,shape and intensity of focused spot,this paper analyzes the effect of astigmatism,coma and spherical aberration with different coefficients on input beam(excitation and depletion)focused spot of STED-4Pi super resolution microscopy.Furthermore,the effect of these three basic aberrations on effective point spread function of STED-4Pi super resolution microscopy is analyzed to evaluate their effect on imaging quality of STED-4Pi super resolution microscopy.The simulation results show that,effected by aberrations,the imaging quality of the STED-4Pi super resolution microscopy degraded.Among them,coma has the relatively large impact on the effective point spread function,resulting in bad imaging quality of STED-4Pi super resolution microscopy.On the other side,specific aberrations can also improve the quality of effective point spread function to a certain extent.Specific astigmatism will improve the resolution of the effective point spread function on the Z axis.Therefore,the imaging quality of STED-4Pi super resolution microscopic improved.Moreover,the image generated by STED-4Pi super resolution microscopy is further denoised.Applying the deep learning-based content-aware image restoration algorithm can denoise the images generated from STED-4Pi super resolution microscopy.According to the calculation of SSIM and subjective evaluation of human eyes,it is found that after processing by this algorithm,better quality images can be obtained.