Synthesis And Application Of Fluorescent Carbon Nanomaterials For Bioimaging

Due to its high sensitivity and high temporal-spatial resolution,fluorescence microscopy represents a key tool to probe molecular interactions in tissue,cell and subcellular organelles in vivo for helping understand the biological process.High quality fluorescence imaging relies strongly on the use of sensitive and phototable fluorescent probes.Compared with traditional fluorophores,fluorescent carbon nanomaterials including carbon dots(CDs)and graphene quantum dots(GQDs)have more superior optical and chemical properties,such as high photostability,excitation-dependent fluorescence,ultra-small size,low toxicity and easy preparation and functionalization,and thus they are attracting great attentions for a wide range of biological applications.In this thesis,we have synthesized a series of fluorescent CDs and characterized their fluorescence properties uisng ensemble and single molecule/particle optical techniques.Based on their unique fluroescence behaviors,we have developed a series of fluorescence imaging methods with high sensitivity and sub-diffraction resolution.The main contents include as follows:(1)We have synthesized highly photostable CDs by chemical oxidation of carbon black with nitric acid.The CDs show robust resistance to photobleaching and they remain constant fluorescence intensity even under 30 min continuous illumination of high-power light source.Further,the anti-bleach fluorescence can be enhanced by plasma resonant silver film,allowing the ultimate signal-to-noise ratio to be increased up to about 30-fold.Taking advantages of silver film-enhanceed anti-bleach fluorescence of CDs and and the easy photobleach ability of cell autofluorescence,we have developed a fluorescence imaging method with high signal-to-noise ratio for cell-targeted imaging.(2)We have investigated the fluorescence properties of CDs at single particle level,and found that they exhibit a burst-like fluorescence blinking behavior.Such blinking has a low duty cycle(?0.003)and high photonoutput(?8000 per switching event),which is comparable to those in Alexa 647 or Cy5 typically used in super-resolution microscopy.Importanly,the CDs show much higher resistance to photobleaching than organic dyes.The stable blinking allows CDs to be used for high-density super-resolution localization imaging with a resolution of 25 nm.The CD-based super-resolution imaging is further demonstrated by rendering CD-stained tubular peptide self-assemblies,CD-packed clusters with well-defined patterns,and CD-stained microtubules in a cell.Finally,this method has been validated as a valuable tool to detect the clustering and distribution of protein receptors in the plasma membrane that are not discerned with normal fluorescence imaging.(3)We have synthesized highly fluorescent N-and O-rich carbogenic small molecular complex(SMC,MW < 1000Da)by using citric acid and ethanediamine as precursors.The SMC exhibits high quantum yield up to 80%,excitation-dependent fluorescence(EDF),and a near-infrared(NIR)emission peaked at 850 nm with an ultralarge Stokes shift(?500nm).SMCs also show strong rRNA affinity,and the resulting EDF enhancement allows multicolor visualization of nucleoli in cells.Furthermore,SMCs can be efficiently accumulated in tumor in vivo after injection into tumor-bearing mice.The NIR emission affords high signal/noise ratio imaging for delineating the true extent of tumor.Importantly,about 80% of injected SMCs can be rapidly excreted from the body in 24 h without no appreciable toxicological responses.