Synthesis Of Fluorescent Blinking Carbon Dots And Their Applications In Super-resolution Imaging

Super-resolution fluorescence(SRF)imaging technology is one of the fastest growing optical imaging methods in recent years.Because it can achieve nanoscale imaging resolution beyond optical diffraction limit,SRF imaging has great potential as a tool for applications from biological sciences,biomedicine to materials science.Currently,the most representative SRF imaging techniques include Stimulated Emission Depletion Microscopy(STED),Photoactivation Localization Microscopy(PALM),Stochastic Optical Reconstruction Microscopy(STORM)and direct STORM(d STORM).Compared with other SRF imaging methods,d STORM can be achieved in the simplest form using spontaneously blinking fluorescent dye with conventional wide-field fluorescence microscope.Such an accessible implementation makes d STORM receive great attentions in a wide research community.To achieve d STORM imaging,the most critical aspect is to find a right fluorescent probe with spontaneously blinking behaviors.So far,there are few fluorescent probes that can be used for d STORM imaging,such as Cy5,AF647 and some of semiconductor quantum dots.Moreover,these fluorescent probes still have great limitations for high quality SRF imaging due to their poor photostability,large size,high duty cycle in blinking and the requirement to bind a largesized biomacromolecules for target recognition.Therefore,the development of fluorescent blinking probes with superior photostability,small size,and even direct recognition of specific target molecules is one of the important research directions and challenges in the field of superresolution imaging.In this thesis,based on fluorescent carbon nanomaterials,we have developed two kinds of carbon dots with superior blinking behaviors,ultra-small size(2-6 nm)and high photostability for d STORM imaging.Furthermore,combined with their physicochemical properties with important applications,we have conducted super-resolution imaging studies for revealing complex processes during performing their specific applications.The main research contents are as follows:(1)Two synthetic methods including strong acid oxidation cutting of carbon black("top" to "down")and citric acid-ethylenediamine dehydration carbonization("down" to "top")were explored to prepare fluorescent carbon nanodots.We obtained ultrasmall-sized single-layer graphene quantum dots and carbon dots with amorphous structure.The physical and chemical structures were then characterized by transmission electron microscopy,atomic force microscopy,Fourier transform infrared spectroscopy,and X-ray photoelectron spectroscopy.Their spectral properties and fluorescence blinking behavior were studied using UV-visible absorption spectroscopy,two-dimensional and three-dimensional fluorescence spectroscopy,and single-particle fluorescence imaging techniques.The results demonstrated that two types of carbon dots possessed significant merits such as low duty cycle,high fluorescence brightness and superior photostability for achieving super-resolution imaging.(2)Combining blinking behavior of graphene quantum dots(GQDs)with their functions as drug carriers,we developed GQDs-super-resolution imaging method to probe drug carrier transportation and delivery process.In this chapter,a quantitative super-resolution algorithm was developed to study the aggregation size of drug carriers on living cell membranes,the number of carriers in aggregates,the distribution of aggregates,ad their links to endocytic process and drug delivery route.The results demonstrated that the uptake efficiency and accumulation of nanocarriers wee controlled by the aggregation and distribution of nanocarriers on the cell surface irrespective of the drug type,and the efficiency of drug delivery depended on the drug’s own loading and release capabilities.(3)Combining blinking behavior of citric acid-ethylenediamine carbon dot(CDs)with their ability to recognize specifically ribosomal RNA(r RNA)in a cell,we developed CDssuper-resolution imaging method to probe the subdiffractional distribution of r RNA in the nucleoli.In this chapter,four types of cells including cervical cancer He La,human glioma U87,mouse melanoma B16,and mouse embryonic fibroblast NIH 3T3 were used as cell models.By using RNase and DNase treatments to cells,we found that the citric acid-ethylenediamine CDs were specifically bound to r RNA and thus recognized fine ultra-structures like dense fibrillar component(DFC)in the nucleolus.Then,the distribution of r RNA in cancer cells and normal cells and the effects of methotrexate,5-fluorouracil,homoharringtonine,and oxaliplatin on the fine structure of nucleolus were studied in detail.The results have demonstrated that CDs-based super-resolution imaging method is a powerful tool to study the mechanism of physiological and pathological function of nucleolar r RNA and the targeted drug design.