Research On The Fabrication And Application Of Single Molecule Localization Imaging Nanoprobes

Among the various microscopy techniques,fluorescence microscopy is one of the most widely used.However,conventional fluorescence microscopy is limited by relatively low spatial resolution which cannot obtain 200 nm because of the diffraction of light.In recent years,super resolution fluorescence imaging that break the diffraction limit has been played an increasingly role in cell biology.Super resolution techniques allow the observation of many subtle biological structures which are not resolvable in conventional fluorescence microscopy.Based on their mechanism of surpassing the diffraction limit,super resolution imaging techniques can be classified into two categories:(I)spatially modulated fluorescence emission and(II)Single molecule localization microscopy(SMLM).Among different super resolution imaging techniques,SMLM including PALM and STORM can provide the highest spatial resolution.However,SMLM requires that the fluorophores must exhibit obvious blinking behavior upon continuous illumination,which limits the kinds of available fluorophores.Usually,blinking behavior is an intrinsic property of fluorescent proteins or specific organic dyes,which can be provoked by strict conditions(at least two lasers for activation and excitation dye molecule,toxicity imaging buffer).Therefore,preparation of fluorescent nanoprobes with excellent biocompatibility and minimal toxicity is prerequisite for expanding the application of SMLM.In this paper,we focus on the preparation of novel fluorescent nanoprobes with single excitation laser and low toxicity.Yolk-shell type nanoprobes for super resolution imaging are presented,the nanoprobes are used to investigate the interaction between nanoparticles(NPs)and cells.The nanoprobes are based on Au@Ag@Si O₂ NPs,yolk-shell structure is formed by etching the inner layer of Si O₂.Then,organic dye molecules are added into the void space of the yolk-shell structure by free diffusion of these molecules.Since the dye molecules can move randomly in the void space,when the molecules move near the metal core of the nanoprobes,the fluorescence is quenched.When the molecules move far away from the metal core,the fluorescence recover.As a result,the molecules in the void space of the nanoprobes can switch between fluorescent state and non-fluorescent state.This fluorescence blinking phenomenon can be used for SMLM.Experimental results confirmed the feasibility of the yolk-shell nanoprobes in SMLM and the nanoprobes are used to investigate the interaction between NPs and cells.The main advantage of nanoprobe include:single excitation laser;non-toxic imaging buffer;suitable for many common general dye molecules and live cell SMLM imaging.Besides,GQDs based dual-labeled FRET nanoprobes with excellent fluorescence intensity blinking behavior suitable for SMLM are presented.In general,the FRET nanoprobes are constructed by attaching Alexa Fluor 488 and Alexa Fluor 568 dye molecules onto the surfaces of GQDs.GQDs serve as the donor and the dye molecules as the acceptor.GQDs are chosen as the donor because of their fine solubility,stable fluorescence,excellent biocompatibility and minimal toxicity.Experimental results confirmed that the FRET effect is observed under single 405 nm laser excitation and the blinking behavior of dual-labeled FRET nanoprobes is much better compared to the single-labeled FRET nanoprobes.Besides,through antibody labeling of the surface of the FRET nanoprobes,dual color SMLM imaging of microtubule in MRC-5 cells is realized.The main advantage of nanoprobes include:single 405 nm excitation laser,non-toxic imaging buffer,suitable for live cell SMLM imaging and the merged image of Alexa Fluor 488 and Alexa Fluor 568 channel show the structure of microtubules more clearly.