Electron microscopy characterization of nanoparticles for biomedical application
Keywords: Materials Science and Engineering.

Over the past two decades, nanotechnology has demonstrated great potential in the field of biology and medicine. Nanomaterials, such as gold nanoparticles, with their superior chemical and physical properties, are widely used in a variety of biomedical research, ways ranging from cancer early detection (e.g. liquid biopsy) to treatment (e.g. hyperthermia therapy). On the other hand, advances in nano characterization techniques have enabled new investigations of naturally occurring nanoscale features in the body, in order to understand the pathological processes associated with them. This dissertation describes the use of advanced electron microscopy to characterize nanomaterials of relevance to the field of medicine. Some nanoparticles are lithographically fabricated, some are chemically synthesized, and others are directly extracted from tissues and cells. The morphological, crystallographic, chemical, optical and other physical properties of these nanoparticles are evaluated using a combination of imaging, diffraction and advanced spectroscopy techniques in a transmission electron microscope (TEM) and scanning electron microscope (SEM). In the first part of this work, surface enhanced Raman scattering (SERS) gold nanoparticles were optimized for sensitive detection of tumors by correlating localized surface plasmon resonances (LSPR) with surface enhancement. Electron beam lithography was used to prototype gold nanostructures with a wide variety of shapes, size, interspacing and in different dielectric environments. The LSPR of these structures were measured using electron energy loss spectroscopy (EELS) in a transmission electron microscope operated in scanning mode (STEM) with monochromation. It is found that nanoparticle size and dielectric environment have the most significant effects on localized surface plasmons, which is collective oscillation modes of the free electron gas at the metal surface. By contrast, interspacing has a weaker influence on surface plasmons for the range studied in this dissertation. Larger nanoparticle size and higher dielectric constant result in lower surface plasmon energies. The novelty of this work is that the LSPR from various nanostructure arrays were correlated with their Raman spectra acquired at different illuminating laser energies after incubation with a Raman dye. It is demonstrated that the largest Raman signal intensities are obtained when the illuminating laser energy coincides with, or is slightly higher than, the gold nanoparticle surface plasmon resonance energies (e.g. 90 nm diameter nanodisc particles with a LSPR energy of 1.94 eV show strongest Raman signal enhancement under a 638 nm (1.94 eV) wavelength laser excitation). By comparing various nanostructure shapes with similar surface plasmon energies, it is shown that sharper nanostructures tend to exhibit stronger surface enhancement. This information is useful in designing nanoparticle combinations to generate the largest SERS enhancement for detection of early stage medical problems such as cancer. The second part of this work is focused on naturally occurring particles, in particular, iron deposits in the hippocampal region of a brain to understand the pathological processes related to Alzheimer's diseases (AD). Recent work on iron accumulation in AD brains has led researchers to hypothesize that the oxidation state of iron may be related to neurodegeneration because ferrous iron, compared with ferric iron, may cause oxidative damage and antioxidant depletion on neurons. First, iron rich regions from AD brain tissues were located using correlative magnetic resonance imaging (MRI), optical microscopy (OM), SEM and energy dispersive spectroscopy (EDS). Cross-sections of tissue containing iron deposits were then extracted using focused ion beam (FIB) and subsequently thinned to make them electron transparent. The relative concentrations of ferric and ferrous ions within the iron deposits were determined by studying the intensity ratios of Fe L3:L2 edges from the energy loss near edge structure (ELNES) of the Fe L edge using monochromated STEM-EELS as above. Massive correlation across biological and physical microscopy and spectroscopy techniques was demonstrated for the first time in this work. These observations and insights provide supporting evidence of ferrous iron as being possibly associated with AD. The third and final section addresses characterization of artificial and natural nanoparticle composites. These hybrid nanoparticles, fabricated via a simple extrusion method, can greatly increase the target specificity and cellular uptake in various biomedical applications such as cancer imaging and drug delivery. A negative staining technique was utilized to provide contrast of biological components of these nanoparticles in TEM, and specific proteins of interest were labeled with antibodies conjugated to 100 nm diameter gold iron oxide nanoparticles (GIONs). The combination of superior magnetic, photonic and other physical properties from artificial nanoparticles, along with cellular specificity and biological compatibility from natural nanoparticles makes these hybrid nanoparticles useful for multi-modality imaging and possible medical treatment. Overall, electron microscopy is a versatile and powerful methodology for characterization of a wide variety of nanomaterials. Advanced microscopic and spectroscopic techniques such as monochromatic STEM-EELS and EDS, which are rarely used in the life sciences, have great potential in bringing unique insight into biomedical research