Interactions Between Fluorescent Molecules and Plasmonic Nanoparticles: A Super-Resolution Study

Single-molecule super-resolution imaging techniques play an increasingly important role in studying complex fine structures, dynamics and interactions in nanoscale confined spaces. The resolution of this technique can be enhanced by plasmonic nanoparticles, yet the complicated interaction needs to be understood in depth. In the present Thesis, I investigate the interactions between fluorescent molecules and gold plasmonic nanoparticles of various structures using three different single-molecule imaging techniques. Chapter I thoroughly introduces the general principles and limitations of single-molecule super-resolution fluorescence microscopy. Plasmonic nanoparticles and how they can be used in plasmon-enhanced single-molecule fluorescence microscopy are also introduced. The interactions between the nanoparticles and fluorescent molecules are complicated and it is essential to understand this interaction at the single-particle single-molecule level. Open questions including what is the distance-dependence of fluorescence enhancement and how is the dye emission mislocalized are also introduced here. The separation distance between the nanoantenna and the fluorescent molecules will drastically influence the effective enhancement. To explore this distance-dependence, in Chapter II, I use PAINT (Points Accumulation for Imaging in Nanoscale Topography) to image the interactions between single fluorescent molecules and a single gold NR. This work demonstrates the great potential of plasmonic nanoparticles to be used in single-molecule super-resolution imaging and indicates that a spacer layer 10 nm is necessary for best enhancement. This distance-dependent enhancement effect is generally applicable to different kinds of probes including cyanine dye molecules and fluorescent proteins. This optimal thickness provides experimental evidence that fluorescent probes labeling membrane proteins which are naturally located within 20 nm from the outer boundary of cells can be enhanced when coupled to an extracellular nanoparticle substrate. We would like to understand this distance-dependent fluorescent enhancement in a structure where the distance between the nanoparticle and the fluorophore is uniform and more precisely controlled. In Chapter III, photoactivatable fluorescent proteins are chemically conjugated to silica coated gold nanoparticles, and their plasmon-enhanced fluorescence properties are carefully studied in terms of separation distance by PALM (Photoactivated Localization Microscopy). This structure avoids the ambiguity in distances existing in PAINT experiments and provides direct evidence for distance-dependent fluorescence enhancement. In addition to fluorescence enhancement, plasmonic nanoparticles also influence other optical properties of nearby fluorescent molecules due to the increased local density of states (LDOS), such as the far-field emission positions. Chapter IV describes a recently discovered phenomenon: the far-field emission detected from a fluorescent molecule coupled to a plasmonic nanoparticle, deviates significantly from the actual emitter position. This deviation, which we call fluorescence apparent emission mislocalization, creates an obstacle for studies of nanoparticle fluorophore interactions, including hot spot mapping, nanostructure morphology reconstruction, and for our purpose, it compromises the accuracy of super-resolution imaging in plasmon-enhanced fluorescence microscopy. In this Chapter, we use a single-molecule study to investigate this effect quantitatively as a function of distance. By developing a new statistical analysis tool, we can resolve the actual emitter positions even from 2D projected images and quantify the mislocalization distance with resolution better than the localization precision.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138681/1/bingf_1.pd