Single-molecule fluorescence (SMF) microscopy is a powerful technique that provides high sensitivity and nanometer-scale resolution for biological imaging. The emission profile of an isolated fluorescent molecule allows the emitter position to be determined on a scale far smaller than the standard diffraction limit of light with a precision that improves with the number of detected photons. While in vitro implementations of SMF have achieved 1.5-nm localization precisions, an outstanding problem in the field is to improve the resolution of SMF imaging in live cells; this has been generally limited to 10 – 40 nm. The main problem is technological: fluorescent proteins (FPs), the genetically encodable labels widely used for biological imaging, are dimmer and shorter lived than the organic dyes employed in vitro. Increasing FP brightness and photostability will significantly improve the precision with which these fluorescent probes are localized in vivo down to a few nanometers, as well as increase average trajectory length for single-particle tracking, and these advances will enable studies of intracellular processes on the molecular scale.
In this Thesis, we use SMF microscopy to characterize fluorescence and attain super-resolution images, and we demonstrate that nanoparticle plasmonics can improve both the brightness and photostability of FPs. The localized surface plasmon mode, or collective oscillation of free electrons, produces a highly enhanced field in the near field of a metal nanostructure. Here, by positioning FPs in the near field of gold nanorods via adsorption or immobilization, we use this coupling to more than double the emission rate of the red FP mCherry, and determine that coupled molecules of the photo-activatable FP PAmCherry emit three times more photons prior to photobleaching. We then extend our methods to in vivo experiments, in which gold nanotriangle arrays serve as extracellular imaging substrates to enhance the emission from membrane-bound proteins in live bacterial cells. Finally, we demonstrate selective excitation of the longitudinal mode of gold nanorods using polarization, and consequently tune the amount of plasmon-enhanced emission observed. The work in this Thesis demonstrates the power of plasmon-enhanced single-molecule fluorescence to strongly impact the bio-imaging field, with implications for human health and disease.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110406/1/donehue_1.pd
