Research On New Methods And Technologies For Realizing Sub-100nm Super-resolution Optical Microscopy

Optical microscopy has been widely recognized as an efficient method for biological and medical observations and investigations,because of its high resolution,real-time observation,minimal perturbation,and convenient operation.During last decades,an international cadre of pioneering researchers have been desperate to develop super resolution optical microscopy and have achieved a lot.Nevertheless,present methods and techniques still have many drawbacks.Among all the super-resolution optical microscopy techniques,point spread function(PSF)engineering and frequency shift are the two basic theories which can break the diffraction limit.This dissertation firstly analyzed these two theories,and proposed two novel methods based them respectively to realize sub-100nm resolution.Based on the PSF engineering,I proposed a novel method called CW-STED differential microscopy.The resolution of CW-STED is decided by the power of the depletion light.Higher power leads to better resolution but more damage to samples.The CW-STED differential microscopy which combines the CW-STED with the subtraction method can enhance both the resolution and SNR of CW-STED.The validity of this method in practice is demonstrated by experiments on both fluorescent nanoparticles and microtubule networks in which a better resolution and contrast than the STED image is achieved.Compared with other resolution enhancement concepts,this method is much simpler and free from using high intensity lasers.Based on the principle of frequency shift,we proposed a novel method,which integrates pattern illumination,the nonlinearity arising from saturation of the fluorophore excited state and Fourier ptychography(FP)algorithm,for super-resolution fluorescence imaging.This method,termed Saturated pattern-illuminated Fourier ptychography(SpiFP)microscopy,could achieve a resolution 4 times that of wide field when the illuminating light intensity approaches the saturation threshold.Increasing light intensity leads to further resolution enhancement.We also make a comparison between SpiFP and saturated structure illumination microscopy(SSIM),and proves that the proposed method exhibit superior robustness to noise,aberration correcting ability,and pattern,s flexibility.This reported method provides an alternative solution of nonlinear pattern illumination microscopy,implying its potential in nanoscale-sized biological observations by wide-field microscopy.