Date of Award
Doctor of Philosophy
In order to elucidate differences between cancerous and analogous normal tissues, nonphotochemical hole burning (NPHB) spectroscopy utilizing MF680 (commonly known as rhodamine 800) was performed. The cationic lipophilic fluorophore MF680 preferentially locates in in situ mitochondria due to the presence of a large negative membrane potential (relative to the cellular cytoplasm) caused by oxidative phosphorylation. The cancerous ovarian (adenocarcinoma, stage 3C) and normal peritoneal tissue samples were surgically removed and characterized at the University of Oklahoma-Health Science Center (Oklahoma City, OK), where the normal tissues were then further categorized as adequate or distant normal, depending on the physical proximity to the cancerous tissue.;The results of these hole burning experiments are interpreted on the basis of the NPHB mechanism and characteristic interactions between the glass-like structured host (in vivo mitochondrial matrix) and the guest (MF680) in the burning of spectral holes, thus providing an image of the cellular ultrastructure. The fluorescence excitation spectra of MF680 in the tissues and the confocal microscopy images of thin sliced tissues incubated with MF680 confirmed the site-specificity of the probe molecules in the cellular systems. A direct comparison of the positions of the MF680 fluorescence excitation peaks between tissues showed only insignificant differences. Hole growth kinetics of MF680 in tissues, unlike the preliminary cultured cell studies, also proved inconclusive in distinguishing the tissue cellular matrices surrounding the chromophores. Measurement of changes in the permanent dipole moment ( f·Deltamu) was accomplished by measurement of changes in hole width in response to the application of an external electric (Stark) field. Stark fields resulted in hole broadening for both tissue samples when laser polarization was either parallel or perpendicular to the applied electric field. The f·Deltamu values for the cancerous tissues were found to be ~1.35 times greater than those of the distant normal analogs on all burn frequencies. The differences in the change in the dipole moments are interpreted in terms of effects from the mitochondrial membrane potential. Results for flow cytometric determination of the mitochondrial membrane potential in the attempt to quantificate f·Deltamu values are also presented.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Matsuzaki, Satoshi, "Hole burning imaging studies of cancerous and analogous normal ovarian tissues utilizing organelle specific dyes " (2004). Retrospective Theses and Dissertations. 1184.