Saturated Absorption Competition Super-resolution Microscopy And Instrument

Abbe diffraction limit has long been regarded as the upper limit of the resolution of optical microscopy,and the invention of super-resolution optical microscopy has successfully bypassed this barrier.Currently,the following mainstream super-resolution techniques are included:stochastic optical reconstruction microscopy,photo-activated localization microscopy,stimulated emission depletion microscopy and structural illumination microscopy.However,there exist some drawbacks for the current super-resolution techniques.The related super-resolution derivatives are hot research topics that it is of great significance to develop universal super-resolution instruments and systems.In this dissertation,super-resolution microscopy based on saturated absorption competition is studied to achieve sub-100-nm resolution imaging in either fluorescent or non-fluorescent realm.By splitting a single-source wavelength pulsed laser,two pulsed beams are obtained,which are modulated temporally and spatally,respectively;On the sample plane,due to the nonlinearity of fluorescence excitation,there occurs fluorescence competition between two pulses,and the obtained point spread function of the temporally-modulated fluorescence is compressed.Finally,the target signal is filtered by a lock-in amplifier to achieve optical super-resolution.Based on the principle of saturation competition microscopy in pulsed mode,the influence of various factors on the imaging results has been discussed.Through a myriad of simulation and experimental works,the reliability and stability of this technology have been verified in super-resolution imaging of nanoparticles and subcellular biological structures.In addition,the optical setup is further improved using a dual-modulation background suppression method,which effectively reduces the Poisson noise caused by the hollow beam.In order to achieve high-resolution three-dimensional saturation competition imaging,the feasibility of either using differential saturation competition in improving the lateral resolution or combining mirror enhancement technology in improving the axial resolution are verified via simulation works.The main content and innovation points of this dissertation include:1.The physical process of saturation absorption competition in pulsed mode is deeply studied,and the competition factor is introduced to analyze the relationship between the signal and the background noise in sub-diffraction region.The influence of a variety of factors on the resulting imaging is simulated and is also verified by experiments,which has guiding significance for the practical imaging.In the aspect of fluorescent imaging,combined with fluorescent nanodiamonds and quantum dots,we for the first time realize dual-color super-resolution imaging with only single laser source based on point-scanning,and obtain long-term imaging with resolution of 1/12 illumination wavelength in biological subcellular structures.At the same time,a novel imaging method of non-fluorescent saturated scattering competition is proposed,which combines gold nanoparticles to achieve ultra-high spatial resolution of 1/13 illumination wavelength.2.A dual modulation method for background suppression is proposed.The generating mechanism of background noise is deeply studied,and the feasibility of dual modulation scheme is verified by simulation and experiment works.At the same time,the influence of the modulation frequencies and demodulation frequency of the two incident beams on the background noise is explored,and the optimal parameters are obtained.The results show that the Poisson background noise of the saturated competition system with dual modulation is lowered by half compared with that of the non-modulated system.3.A three-dimensional differential saturation competition super-resolution method is proposed.By studying the mechanism of differential saturation competition,the optimal subtracting coefficient is obtained,which can achieve high spatial resolution under relatively low competition beam intensity.The results show that the lateral resolution can reach 1/12 illumination wavelength,and the axial resolution can also reach 1/5 illumination wavelength.