Date of Award
Doctor of Philosophy
Kenneth D. Kihm
Anthony English, Seung Joon Baek, Dongjun Lee
The initial goal of this research was to measure the hindered Brownian motion of nanoparticles (100 nm to 500 nm in radii), in varying salinities of water, in order to compare the normal and tangential motion with existing theory. Using techniques developed from this work, brain cancer cells containing vesicles loaded with Nonsteroidal anti-inflammatory drug-Activated Gene-1 (NAG-1), tagged with a Green Fluorescent Protein (GFP), were examined to see what effect the glass cover slip played in hindering their motion.
Several microscopy techniques have been used in this work including Total Internal Reflection Fluorescent Microscopy (TIRFM) and Differential Interference Reflection Microscopy (DICM). TIRFM in is a method used to examine an area approximately 1micrometer from the cover slip, while DICM can be used to examine the coverage area of a cell. Included in this work are several digital image processing techniques that were developed for tracking nanoparticles and biological vesicles, as well as software to examine cellular and focal adhesion coverage area using DICM and Interference Reflection Contrast Microscopy (IRCM), a technique that is especially useful in examining cell substrate interactions.
Results of nanoparticle tracking showed that the tangential motion of the particles followed very closely to the theory proposed by Goldman et. Al., while the normal motion was substantially different than that proposed by Brenner. However it should be noted that Brenner’s theory does not include electrostatic forces that are significant in this work and therefore it should not be concluded that the theory is incorrect. Rather it was concluded that an additional term is needed to account for this added force.
For intracellular vesicle tracking in cancer cells three types of motion were apparent: directional, Brownian, and caged. It was observed that the majority of the motion was either directional or caged and that hindrance values due to hydrodynamic effects were small compared with other hindrance effects. Also a method was established to estimate the average vesicle size base on the observed motion. This method is believed to have potential for use in determining fluid viscosity as well as nanoparticle sizes in future studies.
Margraves, Charles H., "From Nearfield Nanoparticle Tracking To Intracellular Vesicle Tracking. " PhD diss., University of Tennessee, 2008.