Enhanced, Live-Cell, Super-Resolution Imaging and Single-Molecule Emission Spectrum Reshaping

Abstract

Live-cell imaging elucidates subcellular dynamics, and single-molecule imaging extends the capabilities of fluorescence microscopy to the scale of tens of nanometers. To understand the physics of cellular processes on the molecular scale, accurate and precise localization of single molecules is important. The molecule localization precision is related to the brightness of the fluorescence emission, which is sensitive to the local environment. Plasmonic nanoparticles, which act as optical antennas, can enhance the brightness of nearby fluorophores for improved live-cell super-resolution imaging. Additionally, single-molecule fluorescence imaging makes it possible to study light-matter interactions, such as plasmon-enhanced fluorescence, on the nanometer scale. The enhancement of fluorophores is through both a redistribution in the excitation field and a change in the radiative and nonradiative pathways.

In this Thesis, I investigate the effect of the properties of gold nanoparticle arrays, such as particle size, shape and array pitch, on the enhancement factors for plasmon-enhanced live-cell super-resolution imaging, I study the spectral effects of single dyes coupled to individual plasmonic nanoparticles, and I work toward developing an all-fluorescence method for nanothermometry. Chapter I details the background of single-molecule super-resolution fluorescence imaging, plasmon-enhanced fluorescence, and nano-fabrication of plasmonic substrates for fluorescence imaging.

To investigate the live-cell enhancement factors, in Chapter II, I use photoactivation localization microscopy to measure the intensities of single fluorescent proteins in live cells that are imaged on nanosphere lithographed gold nanotriangle arrays of different sizes and pitches. The results of this work demonstrate how fluorescence enhancement depends on the array characteristics and indicate the ability of plasmonic nanoparticle arrays to increase the brightness of a fluorescent protein in living bacteria. The use of plasmonic substrates for enhanced live-cell imaging is generally accessible for membrane-associated targets, and nanosphere lithography is a cheap and easy method for making the plasmonic substrates.

Although we were able to enhance the fluorescence in living cells, we propose to improve the enhancement beyond two-fold by examining a wider range of nanoparticle sizes, nanoparticle shapes, array order, and array pitch. In Chapter II, I present the use of electron-beam lithography to fabricate a wide range of nanoparticle arrays and I measure their optical responses. I use dark-field scattering spectroscopy to measure the resonance strength and spectrum of the nanoparticle arrays, building on conclusions from earlier in Chapter II. Electron-beam lithography allows for tighter control over the particles and arrays for more carefully tuned substrates for enhanced live-cell imaging.

In addition to live-cell enhancement, single-molecule super-resolution imaging enables studies of light-matter interactions. Plasmon-enhanced fluorescence is a distance- and wavelength-dependent process, and super-resolution hyperspectral imaging allows us to study the heterogeneity of coupling. In Chapter III, I study the distance and spectral dependencies of enhanced fluorescence by combining super-resolution imaging with hyperspectral imaging for simultaneous super-localization and spectroscopy. These results demonstrate the power of single-molecule hyperspectral imaging to elucidate subtle changes in the emission spectrum upon plasmon-coupled fluorescence.

In Chapter IV, I present relevant future directions for super-resolution imaging and studies of light-matter interaction such as: single-particle photoluminescence imaging to study the power and temperature dependence of gold nanoparticle photoluminescence, integrating plasmonics and microfluidics for active control of cellular environments with enhanced imaging, and hyperspectral polarization imaging for information dense imaging of many fluorophores in complex environments.