Date of Award
Doctor of Philosophy
Gerald J. Small
Low temperature Nonphotochemical Hole Burning (NPHB) Spectroscopy of the dye rhodamine 800 (MF680) was applied for the purpose of discerning differences between cultured normal and carcinoma ovarian surface epithelial (OSE) cells. Both the cell lines were developed and characterized at the Mayo Clinic (Rochester, MN), with the normal cell line having been transfected with a strain of temperature sensitive Simian Virus 40 Large T Antigen (SV40) for the purpose of extending the life of the cell culture without inducing permanent changes in the characteristics of the cell line. The cationic lipophilic fluorophore rhodamine 800 preferentially locates in in situ mitochondria due to the high lipid composition of mitochondria and the generation of a large negative membrane potential (relative to the cellular cytoplasm) for oxidative phosphorylation.;Results presented for NPHB of MF680 located in the cells show significant differences between the two cell lines. The results are interpreted on the basis of the NPHB mechanism and characteristic interactions between the host (cellular mitochondrial) and the guest (MF680) in the burning of spectral holes, thus providing an image of the cellular ultrastructure. Hole growth kinetics (HGK) were found to differ markedly between the two cell lines, with the carcinoma cell line burning at a faster average rate for the same exposure fluence. Theoretical fits to the data suggest a lower degree of structural heterogeneity in the carcinoma cell line relative to the normal cell line. Measurement of changes in the permanent dipole moment (fDeltamu) were accomplished by measurement of changes in hole width in response to the application of an external electric field (the Stark effect), and found that Deltamu values for the carcinoma line were 1.5x greater than those of the SV40 antigen-free normal analogs. These findings are interpreted in terms of effects from the mitochondrial membrane potential. Results for HGK on the scale of single cells is also presented. Trends observed for large populations of cells (5 x 105 to 2 x 10 6) were concurrently observed for individual cells, with the carcinoma cell line burning at a faster average rate relative to the normal cell line.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Richard Joseph Walsh
Walsh, Richard Joseph, "Nonphotochemical hole-burning imaging studies of in vitro carcinoma and normal cells utilizing a mitochondrial specific dye " (2002). Retrospective Theses and Dissertations. 488.