Fluorescent Blinking Carbon Dots For Super-Resolution Imaging Of Nucleolus

The nucleolus serves as the site of ribosome biogenesis in eukaryotic cells and plays a vital role in a variety of cellular events.The nucleolar dysmorphology is a diagnostic marker in many cancers,and the blockage of nucleolar dysfunction has been regarded as a promising therapeutic strategy.The precise characterizations of nucleolar abnormalities in structure and function require a visualization of nucleolar ultrastructure.The advent of super-resolution（SR）imaging techniques has enabled nanoscopy imaging of the nucleolus ultrastructure beyond the optical diffraction limit.At present,the common fluorophores for SR imaging include organic dye,fluorescent protein and semiconductor quantum dots.The use of these fluorophores for nucleolar imaging,however,is largely restricted by the need of complicated labeling procedures such as immunostaining and gene transfection.Carbon dots（CDs）have attracted increasing attention as a fluorescent probe for bioimaging due to the advantages of good biocompatibility,small size and structural diversity.Recently,some types of CDs have been discovered to show blinking behaviors and binding specificity to the nucleolus,which enables them as a SR probe for nucleolar ultrastructure imaging.Nevertheless,the CDs are complex mixture with heterogeneous structures and photophysics.Those inessential components can interfere with or worsen nucleolus-binding specificity and fluorescent blinking,which greatly limits the application of CDs for nucleolar ultrastructure studies.In this thesis,we separated the CDs by using gel electrophoresis,and identified the positive subtype as a new CD structurally mimicking an RNA-binding protein with fluorescent blinking domains（termed RBP-CDs）,which enables enhancedSR imaging for the nucleolus.By extracting the ultrastructural features of the nucleolus,we demonstrated the CD-depicted nucleolar ultrastructure as a sensitive hallmark to identify and discriminate subtle responses to various stressors.The research content mainly includes:（1）The CDs were synthesized via a“bottom-up”approach by a microwave-assisted reaction of citric acid（CA）and ethylenediamine（EDA）at 180 ^oC for 30 min.The elemental analysis revealed a carbon content of<50%in the CDs,indicating a polymer-like structure.The CDs under scrutiny were separated by the gel electrophoresis into two main subtypes:positive RBP-CDs and negative CDs（N-CDs）.Transmission electron microscopy（TEM）and atomic force microscopy（AFM）images demonstrated that both RBP-CDs and N-CDs were nanoparticles ranging from 2 to 5 nm in size.Analysis using Fourier transform infrared（FTIR）spectra revealed minor differences between them,both containing-NH₂ and-COOH groups.Further analysis by measuring Zeta potentials of the CDs at different p H solutions indicated an isoelectric point（pI）of～10 for RBP-CDs and～3 for N-CDs.Analysis using UV-vis absorption spectroscopy,fluorescence spectroscopy and nuclear magnetic resonance（NMR）spectroscopy demonstrated that both the two CDs exhibited blue fluorescence and binding specificity to the nucleolus by sharing the same chemical groups on particle surface.In addition,RBP-CDs have been demonstrated to contain aromatic domains with red fluorescence excited at 532 nm,which affords high-contrast blinking-basedSR imaging for the nucleolus.Further experiments including fluorescence imaging of nuclease-treated cells and in vitro RNA binding assay confirmed that the CDs could recognize RNA for staining the nucleolus.（2）Fluorescence of single CDs was scrutinized by total internal reflection fluorescence microscopy（TIRFM）.RBP-CDs have much more bright spots than N-PCDs at the same concentration with 532 nm laser excitation.In addition,N-CDs underwent a performant photobleaching in 5 min while RBP-CDs exhibited robust blinking without a decrease in the number of spots in 15 min.The statistical analysis further demonstrated that RBP-CDs have higher photon output and more blinking cycles than N-CDs,which enables high-quality SR imaging.The photophysics resulting in the differences between two types of CDs was also discussed.（3）We rendered the ultrastructure of CD-stained nucleoli by two blinking-basedSR techniques including super-resolution optical fluctuation imaging（SOFI）and direct stochastic optical reconstruction microscopy（dSTORM）.In comparison with N-CDs that only produce an obscure SOFI or noncontinuous dSTORM image,RBP-CDs reveal a ultrastructure of the nucleolus,which is inaccessible by the conventional wide-field（WF）microscopy.Compared with the SOFI image,the dSTORM image allows visualization of more details of the nucleolus with less background.Moreover,a stack of 5000 frames were sufficient for rendering a SR image.By using RBP-CDs,we cannot only depict the morphology and size of the nucleolus,but also can extract the ultrastructure features that reflect the distribution and density of RNA and the distribution of regions lacking RNA in the nucleolus.（4）We used RBP-CDs to stain different types of cells including human cell lines（human embryonic kidney 293A,glioblastoma U87 and lung cancer A549 cells）and mouse cell lines（normal CHO,murine breast cancer 4T1 and colorectal cancer CT26 cells）.The CD-rendered high-definition image provides a great deal of ultrastructural details for the nucleolus,and in combination with the t-distributed stochastic neighbor embedding（t-SNE）dimensionality reduction method,allows visualization and accurate distinguishment of different cells from the same cell types.Furthermore,we demonstrated that the CD-depicted ultrastructure could serve as a sensitive hallmark for sensing and discriminating nucleolar stress responses under cell malignancy and various stressors.We envision that RBP-CDs will become a broadly useful probe for nucleolar stress studies in cell diagnostics and therapeutics.