Non-fluorescent Super-resolution Bio-microscopy Based On Nonlinear Saturation Scattering Of Gold Nanoparticles

Optical microscopy plays an important role in the development of microbiology,and has become an indispensable tool in other fields such as materials science,medicine,optics,and energy.Since the development of the first modern microscopic imaging system in the 16^th century,optical microscopic imaging systems have been restrained by optical diffraction limit,preventing researchers to observe the biological fine structures below 200 nm.In recent decades,with the continous improvement of related optical technology and materials research,more and more super-resolution microscopic imaging techniques that break through the diffraction limit have been proposed.The2014 Nobel Prize in Chemistry was awarded to three distinguished scientists who invented super-resolution imaging techniques,which further promoted super-resolution technology.Compared with other super-resolution imaging techniques,stimulated emission depletion microscopy is highly compatible with confocal microscopy systems.The combination of Gaussian-shaped excitation and doughnut-shaped depletion beams is used to realize super-resolution imaging.The advantage is that the super-resolution image of the sample can be obtained directly without further imaging post-processing.Gold nanoparticles are widely used in biological imaging,immunolabeling,photothermal therapy,drug carriers,etc.,and play a very important role in the field of biological research.Here,this thesis starts from the traditional STED microscopy technology,analyzes the challenges confronted.To tackle these challenges,we propose non-fluorescence super-resolution imaging techniques based on the nonlinear saturation scattering properties of gold nanoparticles.Finally,its application in multiplexed super-resolution imaging of biological samples is demonstrated.The details are as follows:1)Construction of a non-fluorescent STED super-resolution microscopy imaging system.Considering the role of the nonlinear characteristics of plasmonic nanoparticles in experiments,pulsed beam was used as the excitation source,and continuous-wave beam was used as the depletion source.Based on a confocal microscope,a non-fluorescent super-resolution microscopic imaging system was successfully constructed by introducing a dual-beam approach.Finally,a 40-nm resolution was successfully obtained by performing stimulated emission depletion imaging based on the fluorescent sample.2)Study on nonlinear saturation scattering effects and super-resolution imaging of different gold nanoparticles.The surface plasmon effect of gold nanoparticles has strong size and shape dependence.Through the study of the nonlinear characteristics of gold nanoparticles,we demonstrate that the saturation scattering characteristics of gold nanoparticles are correlative with surface plasmon effects and all-optical modulation characteristics.As the size of the gold nanoparticles increases,the surface plasmon resonance peak appears"red shift".For the saturation scattering characteristics of gold nanoparticles,the plasmonic effect plays a major role,that is,the closer the plasmon resonance to the wavelength of the modulated light,it is easier for gold nanoparticles to reach saturation,and the all-optical switching has stronger modulation strength.According to the experiment,Gaussian excitation and donut-shaped modulation beams were used to illuminate gold nanoparticles of different sizes.The imaging resolution reaches 83 nm,which is one seventh of the wavelength,about three orders of magnitude reduced inhibition intensities compared with the conventional STED system,which is of great significance for further applications to scattering based non-fluorescence super-resolution microscopy systems in biological environments.3)Scattering type non-fluorescent multiplexed super-resolution imaging.Based on the aforementioned studies on the saturation scattering properties and super-resolution imaging of gold nanoparticles,we demonstrate that gold nanospheres of different sizes can be used as multiplexed super-resolution probes in human biological osteoblasts?MG-63?.The combination of the all-optical modulation characteristics and size effect is further studied.For the first time,the two-color multiplexed super-resolution biological imaging based on the saturation scattering characteristics of gold nanoparticles of is achieved.The imaging resolution reaches 120nm.In addition,there are three orders of magnitude reduction in the suppression of optical powers,which is of great significance for applications to scattering based non-fluorescence super-resolution microscopy systems in biological complex environments.The innovation of this thesis is to systematically study the saturation scattering properties of gold nanoparticles,and propose and construct a multiplexed non-fluorescent super-resolution microscopic imaging system based on the saturation scattering characteristics of gold nanoparticles.The application to biological samples to obtain a two-color multiplexed super-resolution imaging based on gold nanoparticles of different sizes is demonstrated at an ultralow suppression laser power,nearly three-order of magnitude reduced compared to the state-of-the-arts of STED nanoscopy.