Fluorescent Blinking Carbon Dots For Quantitative Super-Resolution Imaging

The last decade has seen impressive progress in the development of super-resolution fluorescence microscopy that enables us to uncover subcellular structures at nanoscale resolution.Among them,single molecule localization microscopy(SMLM)such as photoactivated localization microscopy(PALM)and stochastic optical reconstruction microscopy(STORM)have attracted great attention because they provide near-molecular resolution and allow quantitative analysis of molecular interactions and organization phenomena in cells such as protein oligomers,clusters,and their distributions within a diffraction spot.The challenges,however,lie in spontaneous on/off blinking and photobleaching recovery of fluorophores,which readily lead to overcounting artifacts.Also,undercounting can arise from spatio-temporal mixing of localizations from multiple molecules in the case of high photoactivation power,fast photoactivation schemes,or blinking events with long off-times.To date,a technique that allows accurate molecular counting with readily accessible suitable chromophores and standard fluorescence microscopes is not readily available.In this thesis,by using small CDs,we explore and manipulate blinking behavior of nanoparticles with diverse structures and surfaces.Most importantly,we develop a qSMLM method based on the CD blinking to differentiate and count single molecules at nanoscale resolution.We demonstrate that this qSMLM has a high counting accuracy of>97%at a molecular density of 500 per(?)m~2.As an example with immediate biological relevance,we use the qSMLM to scrutinize G-protein coupled receptor(GPCR)oligomerization and clustering on the cell membrane.The research content mainly includes:(1)Three types of CDs,including graphite(or graphene),amorphous and polymer-like particles,were synthesized via the"bottom-up"and"top-down"strategies by using a variety of carbon sources as precursors.The physical and chemical structures of the CDs were characterized by using transmission electron microscopy,atomic force microscopy and Fourier transform infrared spectroscopy.The optical properties of the CDs were studied by using UV-visible absorption spectroscopy,two-dimensional and three-dimensional fluorescence spectroscopy.The results demonstrate that the three types of carbon dots have particle diameters ranging between 2-10 nm,and they exhibit regulable fluorescence emission by controlling their structure and surfaces.(2)Fluorescence of single CDs were studied by total internal reflection fluorescence microscopy and confocal-type fluorescence microscope with an avalanche photodiode detector,which confirmed spontaneous fluorescent blinking for the three types of CDs.We subsequently explored the effects of the CDs’structure,size,surface and ambient environment(e.g.air,water,buffer and p H)and imaging conditions(e.g.excitation wavelength,power,and exposure time)on fluorescent brightness and blinking characteristics.The results demonstrated that the localization accuracy and blinking time,which served as two key parameters for super-resolution imaging,allowed to be regulated by controlling the CD type and imaging conditions.(3)Based on the blinking characteristics of CDs,we developed a new algorithm for grouping all localizations from an identical particle into single localization,which enabled accurate molecular counting of multiple particles within a diffraction spot.By simulation and transmission electron microscopy imaging of CDs-labeled peptide assemblies,we demonstrated that the CD-based qSMLM method had a counting accuracy of>97%at a molecular density of 500 per(?)m~2.(4)Using this CD-based qSMLM method,we achieved super-resolution localization imaging of CD-labelled chemokine receptor CXCR4 on a cell membrane.Combined with three spatial analysis methods including noisy density-based clustering analysis(DBSCAN),Ripley’s k function,and pair correlation Function(PCF),we scrutinized the oligomerization and clustering of CXCR4 and their distribution patterns upon the treatments of agonist(SDF-1)and inverse agonist(TC14012).In addition,we monitored the alterations of CXCR4 distributions by the treatment of methyl-β-cyclodextrin(MβCD)to disorganize lipid raft microdomains,which demonstrated the presence of CXCR4 clusters on a cell membrane.This work promises that CD-based qSMLM will become a powerful tool to broadly study sub-diffraction structures,thereby allowing access to the organization at the molecular level.